Cancer cachexia in STK11/LKB1-mutated non-small cell lung cancer is dependent on tumor-secreted GDF15

Domain: neuroscience
Published: 2026-01-30
Editorial outcome: major_revision

Abstract
Cachexia is a wasting syndrome involving adipose, muscle, and body weight loss in cancer patients. Tumor loss-of-function mutations in STK11/LKB1, a regulator of AMP-activated protein kinase, induce cancer cachexia (CC) in preclinical models and are linked to weight loss in non-small cell lung cancer (NSCLC) patients. This study examines the role of the integrated stress response (ISR) cytokine growth differentiation factor 15 (GDF15) in regulating cachexia using patient-derived and engineered STK11/LKB1-mutant NSCLC lines. Tumor cell-derived serum GDF15 levels are elevated in mice bearing these tumors. Treatment with a GDF15-neutralizing antibody or silencing GDF15 from tumor cells prevents adipose/muscle loss, strength decline, and weight reduction, identifying tumors cells as the GDF15 source. Restoring wild-type STK11/LKB1 in NSCLC lines with endogenous STK11/LKB1 loss reverses the ISR and reduces GDF15 expression rescuing the cachexia phenotype. Collectively, these findings implicate tumor-derived GDF15 as a key mediator and therapeutic target in STK11/LKB1-mutant NSCLC-associated cachexia.

Introduction
An understanding of the tumor and host genetic drivers of cancer cachexia (CC) remain elusive. This knowledge would be relevant from both a biomarker and therapeutic perspective. To overcome some of these knowledge barriers, we previously reported findings from a screen of human NSCLCs assessing their capacity to induce cachexia-associated fat and muscle wasting in immunodeficient mice1. We dichotomized a cohort of patient-derived NSCLC lines with CC-inducing capacity in vivo and a matched cohort of NSCLC lines without CC-inducing capacity. Interestingly, 80% of the CC-inducing NSCLCs possessed loss-of-function variants in STK11/LKB1, the upstream regulator of the cellular energy sensor AMP-activated protein kinase (AMPK). We subsequently showed that STK11/LKB1 is a driver of cachexia development by silencing its expression in patient-derived and mouse tumor lines and demonstrating host wasting after subcutaneous transplantation into immunocompromised and syngeneic mice, respectively1. Others have shown that STK11/LKB1 silencing in genetically engineered Kras mutant mice induce CC in a majority of mice2. Finally, in a cohort of nearly 250 advanced NSCLC patients, ctDNA-based liquid biopsy assays established a significant correlation between a patient having a tumor with STK11/LKB1 functional variant and the presence of CC-associated wasting at cancer diagnosis1.

Having shown that tumor STK11/LKB1 loss-of-function mutation is not only a predictive and prognostic biomarker but also functionally involved in CC induction/progression, we sought to identify the downstream tumor-intrinsic molecules transducing mutant STK11/LKB1 CC signals. A candidate approach evaluating tumor-secreted factors with the potential to influence host wasting that had a dependence on tumor STK11/LKB1 functional loss led us to investigate GDF15 as such a molecule. GDF15 is an inflammatory cytokine of the TGF\beta superfamily with many pathophysiologic roles, including in metabolism where it is thought to promote anorexia and subsequent weight loss by direct signaling of the hindbrain through the glial-cell-derived neurotrophic factor family receptor \alpha-like (GFRAL)3–10. Originally shown to be released after activation of the integrated stress response from tissue injury or nutrient deprivation, GDF15 has now been found to regulate a number of fundamental processes11–13. Secreted GDF15 is first produced as a pro-form of protein within cells and only after processing by proteases is it converted into a mature form that can be found in systemic circulation acting centrally on GFRAL-positive neurons in the hindbrain13–15. Systemically, GDF15-induced anorexia was previously thought to be relevant when triggered by platinum-based and other cytotoxic chemotherapies offered to cancer patients16,17. Elevated circulating concentrations of GDF15 are assumed to be a consequence of host tissue response to patient exposure to these cytotoxic agents or as a result of disease progression16. Recent translational investigations also support an association between elevated GDF15 serum levels and the development of NSCLC-associated and other cancer-associated cachexia18–20. Independent of central effects, there are now other suggestions that GFD15 may signal peripherally by regulating contractility and energy expenditure in skeletal muscle21,22. In addition, antibody/small molecule inhibitors of GDF15/GFRAL signaling are being assessed in clinical trials to block cancer-associated cachexia16,23,24. Whether circulating GDF15 primarily originates from tumor or host cells, and the dependencies of tumor systems on GDF15 action remain unknown.

Here, we took advantage of our previous efforts to characterize the cachexia-inducing potential of a cohort of patient-derived NSCLC lines to delineate a tumor’s GDF15 dependence in inducing cachexia and in identifying tumor intrinsic (human) versus extrinsic (murine host) contributions to systemic GDF15 concentrations. Leveraging our well-annotated cohort of human NSCLC patient-derived xenografts with and without STK11/LKB1 variants, we establish an association between tumor cell GDF15 mRNA expression, elevated circulating tumor-derived GDF15 levels, and loss-of-function STK11/LKB1 variants. Parallel use of GDF15 neutralizing antibody and tumor cell GDF15 silencing in multiple STK11/LKB1-mutated patient-derived and murine Kras/Trp53/Stk11 NSCLC lines transplanted subcutaneously or orthotopically into murine models abrogated lung cancer cachexia development. Reconstitution of wild-type STK11/LKB1 into a STK11/LKB1-mutated patient-derived NSCLC line decreased the tumor cell integrated stress response resulting in reduced GDF15 expression, processing, and release; suppressing the CC phenotype. Collectively, our data supports the role of tumor-secreted GDF15 in mediating mutant STK11/LKB1 NSCLC cachexia development.

Results
Characterization of GDF15 tumor and circulating levels across a NSCLC cachexia cohort
We previously identified 17 human non-small lung cancer (NSCLC) lines that produced an unambiguous phenotype of cachexia (n=10) or non-cachexia (n=7) when injected subcutaneously into immunodeficient mice1. Of the 10 lines that induced host wasting, 8 lines had genetic variations in STK11/LKB1 with moderate to high predicted functional impact. In a candidate approach to identify GDF15’s relevance to STK11/LKB1-mediated cachexia in NSCLC, we assessed tumor GDF15 mRNA expression and systemic GDF15 serum concentrations in the context of host wasting in the cachexia (red) and non-cachexia (black) lines from this cohort (Fig. 1A). GDF15 gene expression was assessed by quantitative real-time PCR (RT-PCR) using human (tumor cell-derived) and mouse (host cell-derived) primers to distinguish tumor cell and host cell changes, respectively. Expression of human GDF15 mRNA was significantly elevated in a majority of the CC-inducing lines compared to non-cachexia-inducing lines (Fig. 1B), with the exception of the H1395 and H1993 cachexia-inducing lines that had comparable levels to the non-cachexia cohort. Mouse Gdf15 mRNA expression in tumors, representing host cell contribution to tumor expression, was statistically unchanged between the cachexia and non-cachexia groups (Fig. 1C).

To evaluate serum concentrations of GDF15 from the mice transplanted with the human cancer lines, we first established human (Supplementary Fig. S1A) and mouse (Supplementary Fig. S1B) specific GDF15 ELISA s that only recognized human GDF15 (tumor cell-intrinsic source) and murine GDF15 (host cell source), respectively. Serum concentrations of human GDF15 were significantly elevated in mice injected with the cancer cachexia-inducing STK11/LKB1-mutated NSCLC lines compared to the non-cachexia wild-type STK11/LKB1 NSCLC lines and the PBS control cohort (Fig. 1D), whereas the serum mouse GDF15 concentration was statistically unchanged between the cachexia, non-cachexia, and PBS control groups (Fig. 1G). Importantly, there was a strong correlation between tumor human GDF15 mRNA expression and serum human GDF15 concentrations (Fig. 1E). By contrast, there was no significant association of tumor mouse Gdf15 mRNA expression with serum mouse GDF15 concentrations (Fig. 1H). Finally, there was no significant correlation between serum human GDF15 levels and tumor volumes from the in vivo studies (Fig. 1F). Overall, this data suggested that loss of functional STK11/LKB1-driven NSCLC cachexia is correlated with increases in GDF15 mRNA expression and serum concentrations that are derived from the tumor cells and not the host.

Human STK11/LKB1-mutant NSCLC dependence on high circulating GDF15 concentrations for cachexia induction
We next sought to identify if NSCLCs capable of inducing cachexia secondary to STK11/LKB1 loss were dependent on elevated circulating GDF15 to induce wasting. More specifically to establish that tumor cell-secreted GDF15 initiates cachexia induction, the H1573 NSCLC cell line was transduced with lentiviral CRISPR/Cas9 constructs containing guide RNA targeting GDF15 (H1573\DeltaGDF15) to silence expression. We used the STK11/LKB1 mutant, CC-inducing patient line H1573 since it exhibited the highest tumor GDF15 mRNA expression (see Fig. 1B) and the second highest serum concentration of tumor-derived GDF15 (see Fig. 1D) when xenotransplanted subcutaneously into immunocompromised mice. As a control, the parental H1573 line was also infected with guide RNA directed against GFP (H1573\DeltaGFP). Immunoblot analysis of the engineered H1573 cells determined that GDF15 was only silenced in the H1573\DeltaGDF15 tumor cells compared to the parental H1573 and the H1573\DeltaGFP tumor cells (Fig. 2A). These three lines and a PBS vehicle control were subsequently injected subcutaneously into NOD/SCID mice followed by measurement of tumor growth, food intake, body composition, body weight, and grip strength over time. As shown in Fig. 2B, C, tumors derived from the parental H1573, H1573\DeltaGFP, and H1573\DeltaGDF15 cell lines showed similar tumor growth kinetics and tumor weights at sacrifice, respectively. Immunoblot analysis verified that tumors derived from the H1573\DeltaGDF15 line were durably suppressed from expressing GDF15 (Fig. 2A). Compared to mice harboring parental H1573 or H1573\DeltaGFP tumors demonstrating serum GDF15 concentrations of ~4ng/ml, serum from mice xenotransplanted with H1573\DeltaGDF15 tumors had undetectable human (tumor-derived) GDF15 concentrations at levels comparable to findings in non-tumor bearing PBS-injected mice (Fig. 2D). ELISA measurements also revealed no significant changes in the serum mouse (host cell-derived) GDF15 concentrations in parental or engineered lines compared to the PBS control (Fig. 2E), suggesting a lack of host contribution to serum GDF15 levels.

Critically, compared to the mice injected with parental H1573 or H1573\DeltaGFP cells, mice injected with the H1573\DeltaGDF15 cell line had a rescue of their body weight (Fig. 2H), tumor-free body weight (Fig. 2I), lean mass (Fig. 2K), tumor-free lean mass (Fig. 2L), and fat mass (Fig. 2J), similar to levels observed in the PBS-injected, non-tumor bearing mice. Mice bearing STK11/LKB1-mutated NSCLC tumors with intact GDF15 (parental 1573 and H1573\DeltaGFP) demonstrated a significant decrease in grip strength, whereas mice bearing H1573\DeltaGDF15 tumors had similar grip strength to PBS-injected non-tumor bearing mice (Fig. 2M). Measured another way, we provide in tabular form the ratio of grip strength to experiment day 0 body weight to generate a relative grip strength per mouse in each cohort (Supplementary Data 1).

Silencing of GDF15 promoted minimal change in food intake in mice compared to the mice injected with parental H1573 or H1573\DeltaGFP cells (Fig. 2G). At first approximation, this finding was counter-intuitive to the function that GDF15 has been reported to have a role in regulating food intake. However, from a review of the literature, a lack of food intake change in GDF15-dependent cancer cachexia models was also observed by other investigators23. This group also failed to show increased food intake with suppression of GDF15-GFRAL signaling or significant suppression of food intake with elevated GDF15 serum concentrations. Earlier pre-clinical work by our group found that there are decreased levels of host systemic leptin to maintain food intake in the setting of tumor-secreted cachexia factors that typically decrease food intake and cause adipose wasting25. We next measured serum leptin concentrations to identify if this suggested compensation mechanism for food intake is observed in the GDF15-dependent H1573 cancer cachexia model. Figure 2F demonstrated that in control conditions of only PBS administration or with H1573\DeltaGDF15 cell administration, tumor bearing mice had baseline serum leptin concentrations corresponding to the presence of normal adipose levels. In the cohorts administered the 1573 parental or H1573\DeltaGFP control cells, leptin concentrations had decreased significantly. The host compensation mechanism of decreasing serum leptin provides a possible explanation of why there are no food intake changes observed in this GDF15-dependent cancer cachexia model.

From a tissue perspective, there was protection against loss of both epididymal (Fig. 3A) and inguinal (Fig. 3B) white adipose tissue (eWAT and iWAT) mass in mice injected with H1573\DeltaGDF15 cells as compared to mice injected with parental H1573 or H1573\DeltaGFP cells. Preservation of host adipose tissue with tumor cell GDF15 silencing was further verified by H&E staining of epididymal white adipose tissue (Fig. 3C) and by measurement of average (Fig. 3D) or distribution of (Fig. 3E) adipocyte cross-sectional areas showed similar adipocyte sizes to PBS-injected mice and significant larger adipocytes compared to those adipocytes taken from mice harboring parental H1573 or H1573\DeltaGFP tumors. Both gastrocnemius (Fig. 3F) and quadriceps (Fig. 3G) weights were preserved similar to PBS-injected mice levels with tumor cell silencing of GDF15. Laminin staining of the gastrocnemius muscle revealed a significant increase of the total fiber cross-sectional area of the mice with the H1573\DeltaGDF15 tumors compared to mice harboring the parental or H1573\DeltaGFP tumors (Fig. 3H, I). Finally, transcript levels of gastrocnemius and soleus Fbxo32 and Trim63 genes, both part of ubiquitin ligase complexes relevant to muscle atrophy26, were negatively correlated with mouse body weight – i.e., their mRNA expression was elevated in mice bearing parental H1573 or H1573\DeltaGFP tumor while being low in mice bearing H1573\DeltaGDF15 tumors or mice administered PBS - providing an explanation for the reduced muscle loss in this experimental arm of the study (Fig. 3J and Supplementary Fig. S2). Recently, a group highlighted the role of sarcolipin (Sln) in supporting GDF15 induced, food intake-independent body weight loss by regulating energy expenditure in skeletal muscle22. We, therefore, next evaluated the mRNA levels of muscle Sln in the H1573 cancer cachexia model. Gastrocnemius (Fig. 3J) and soleus (Supplementary Fig. S2) muscle mRNA levels of Sln expression were negatively correlated with mouse body weight. Muscle Sln was elevated in mice bearing STK11/LKB1-mutated tumors with intact GDF15, in cohorts of parental H1573 or H1573\DeltaGFP tumor implanted mice. Muscle Sln expression remained low in mice bearing H1573\DeltaGDF15 tumors or mice administered PBS, providing an explanation for food intake-independent body weight loss in the GDF15 dependent H1573 cancer cachexia model. Overall, these studies demonstrated that STK11/LKB1-mutated H1573 NSCLC intrinsically rely on tumor cell-derived GDF15 for promotion of cachexia-associated adipose and muscle atrophy.

To identify any sexual dichotomy in cancer cachexia mechanisms and outcomes, we next conducted the same studies as depicted in Fig. 2 in the setting of female immunodeficient mice. Again, the H1573 NSCLC line was transplanted into mice unadulterated (parental control), with GFP silencing as a control (H1573\DeltaGFP), or with GDF15 silencing as an experimental approach (H1573\DeltaGDF15). Though tumors grown in the mice transplanted with the H1573\DeltaGDF15 line were on a slower growth trajectory than controls (Supplementary Fig. S3A), their final weights at sacrifice were comparable to controls and no longer statistically different (Supplementary Fig. S3B) in the female mice. Similarly to the findings in male mice, human serum GDF15 was suppressed to background levels compared to controls in female mice bearing H1573\DeltaGDF15 tumors (Supplementary Fig. S3C), with no significant level of mouse GDF15 measured in the serum of any cohort (Supplementary Fig. S3D). Phenotypically, GDF15-silenced H1573 tumor bearing mice demonstrated a preservation of body weight (Supplementary Fig. S3G), fat mass (Supplementary Fig. S3H), lean mass (Supplementary Fig. S3I), and grip strength (Supplementary Fig. S3J and Supplementary Data 1). From a tissue perspective, epididymal white adipose tissue (Supplementary Fig. S3K), inguinal white adipose tissue (Supplementary Fig. S3L), gastrocnemius (Supplementary Fig. S3M) and quadriceps (Supplementary Fig. S3N) tissue weights were also preserved in mice bearing H1573\DeltaGDF15 tumors vs control H1573 tumors. As conserved in male and female tumor cachexia murine models, we observed no decrease of food intake in the female mice bearing parental H1573 or H1573\DeltaGFP tumors compared to PBS administrated mice and no increased food intake in mice bearing H1573\DeltaGDF15 tumors compared to the multiple cohorts of mice displaying cachexia (Supplementary Fig. S3F). The serum leptin concentrations decreased in mice with cachexia and remained stable in mice without cachexia as depicted in Supplementary Fig. S3E, again providing a possible explanation of why there was no net food intake changes observed in this GDF15-dependent cancer cachexia model leveraging female mice, paralleling findings from the 1573 experiment performed with male mice (see Fig. 2F). In summary, our cachexia findings were consistent across male and female mice, showing no obvious sexual dichotomy.

Orthotopic modeling of STK11/LKB1-mutated NSCLC recapitulates the cachexia phenotype
We next conducted parallel studies with isogenic H1573 NSCLC lines by administering them orthotopically into the parenchyma of the left lung of immunocompromised mice as previously described27–29. Animals were administered either PBS, H1573\DeltaGFP cells, or H1573\DeltaGDF15 cells in Matrigel and permitted to form foci of tumors in vivo as depicted in Fig. 4A. There was no difference in the ratio of tumor to lung area in H1573\DeltaGFP or H1573\DeltaGDF15 tumor-bearing mice (Fig. 4B). As predicted, the mice bearing H1573\DeltaGFP lung tumors demonstrated elevations in circulating human GDF15 compared to PBS- or H1573\DeltaGDF15-bearing mice (Fig. 4C). There was no murine GDF15 in circulation in any of the cohorts (Fig. 4D). As demonstrated in Fig. 4E, the H1573\DeltaGDF15-bearing mice did not demonstrate an increase in food intake when compared to the control cohorts. However, the H1573\DeltaGFP tumor-bearing mice had a significant decrease in systemic leptin concentrations compared to the H1573\DeltaGDF15 tumor-bearing mice (Supplementary Fig. S8A), once again providing a possible explanation for the lack of change in food intake. All these findings translated into mice with H1573\DeltaGDF15 orthotopically placed lung tumors being protected against cachexia-associated body weight change (Fig. 4F), fat mass loss (Fig. 4G), and lean mass loss (Fig. 4H) compared to control cohorts. With respect to this last metric, silencing of GDF15 in these lines suppressed quadriceps and gastrocnemius tissue weight loss (Fig. 4J) resulting in a preservation of grip strength (Fig. 4I and Supplementary Data 1) when compared to mice injected with control tumor cells or PBS alone. Staining of gastrocnemius tissue (Fig. 4L) and myocyte cross-sectional area evaluation (Fig. 4K) further demonstrated the maintenance of larger myocytes with GDF15 silencing similar to PBS-injected mice and significantly increased compared to H1573\DeltaGFP-injected mice. With respect to more granular review of the adipose, the H1573\DeltaGDF15-bearing experimental mice were protected against epididymal and inguinal white adipose tissue loss in addition to protection from brown adipose tissue loss based on terminal tissue weight measurements (Fig. 4M). Sectioning and staining of epididymal white adipose tissue by H&E verified preservation of cell size, numbers, and architecture when GDF15 was silenced (Fig. 4L), resulting in preservation of adipocyte cross-sectional area similar to PBS-injected mice and significantly increased compared H1573\DeltaGFP-injected mice (Fig. 4N). In total, we found that placement of cells to create tumor subcutaneously or orthotopically in the lungs solicited near identical cachexia development, and associated changes to muscle and adipose. These findings are suggestive of the importance of tumor-secretory factors in regulating cachexia development from a para/endocrine approach irrespective of anatomic location.

Antibody directed against GDF15 suppresses cancer cachexia-associated adipose and muscle wasting
The antibody against GDF15 (Pfizer murine analog to ponsegromab) used in our subsequent studies is reported to neutralize both human and murine orthologs30. As a control experiment, we showed that serial administration of the GDF15 antibody itself had no effect on wild-type mice with respect to changes in relative body weight (Supplementary Fig. S4A), fat mass (Supplementary Fig. S4B), and lean mass (Supplementary Fig. S4C), demonstrating nearly identical curves to mice receiving PBS or non-specific IgG. Subsequently, to continue testing the importance of tumor and/or host GDF15 to STK11/LKB1-associated NSCLC cachexia, we used a pharmacologic “prevention” approach by treating H1573 tumor-bearing mice with either vehicle (none), non-specific IgG monoclonal antibody, or the neutralizing antibody against GDF15 before any evidence of cachexia wasting and administered every 7 days over the span of the study (Fig. 5A). Another control in the experiment included a mouse cohort bearing H1573\DeltaGDF15 tumors, a model we verified above that cannot induce cachexia in vivo due to a lack of tumor-secreted GDF15. Administration of GDF15 neutralizing antibody or control IgG antibody had no effect on H1573 tumor growth (Fig. 5B) or weight (Fig. 5C) compared to parental H1573 or H1573\DeltaGDF15 tumor-bearing mice. Human ELISA analysis of serum demonstrated that the administration of GDF15 antibody significantly neutralized the serum human GDF15 concentrations from ~5ng/ml in the vehicle- and IgG-treated H1573 mouse cohorts to ~0ng/ml in mice (Fig. 5D), only matched in absence of circulating levels in mice bearing H1573\DeltaGDF15 tumors. Mice xenotransplanted with H1573 and administered GDF15 neutralizing antibody had a preservation of tumor-free body weight (Fig. 5E), fat mass (Fig. 5G), or tumor-free lean body mass (Fig. 5F) similar H1573\DeltaGDF15-bearing mice and significantly increased compared to vehicle- and IgG-treated H1573 tumor bearing mice. Again, non-specific IgG antibody treated mice were observed with lower serum leptin concentrations and GDF15 antibody administrated mice were observed with normal/baseline leptin concentrations (Supplementary Fig. S8B), providing a possible explanation for the lack of change in food intake. Overall, treatment with the GDF15 neutralizing antibody dramatically suppressed the development of cachexia-associated fat/lean mass and body weight loss normally induced by the STK11/LKB1-mutant H1573 NSCLC line.

We next conducted a cachexia “rescue” experiment by determining if GDF15 neutralizing antibody administration could stop and reverse cachexia associated wasting once muscle and fat losses had already been established. In this study, four cohorts of mice were employed – a control group administered only PBS, and three H1573 parental cohorts administered nothing, non-specific IgG antibody, and the GDF15 antibody. But, in this study, antibody or vehicle was only administered serially after 20% fat mass loss had been proven by longitudinal ECHOMRI (Fig. 5H). As anticipated, there was no difference in tumor growth kinetics (Fig. 5I) or weights (Fig. 5J). ELISA verified that the mice bearing H1573 tumors administered GDF15 antibody had a reduction in serum GDF15 to levels approaching non-tumor bearing control mice (Fig. 5K). Overall, GDF15 antibody administration even after the development of fat loss as a “rescue” strategy returned H1573-tumor bearing mice body weight loss (Fig. 5L), fat mass (Fig. 5M), epididymal white adipose weight loss (Fig. 5N), lean mass (Fig. 5O), and gastrocnemius tissue weight (Fig. 5P) back to levels similar to mice bearing no tumor and only administered PBS. Hence, it is apparent that the GDF15 neutralizing antibody can be used for prevention and/or rescue of cachexia induced by NSCLCs with STK11/LKB1 loss of function alterations promoting wasting through tumor GDF15 induction.

Human STK11/LKB1-mutant NSCLC dependence on intermediate and low circulating GDF15 concentrations for cachexia induction
Having established that mice xenotransplanted with human NSCLC lines demonstrating high circulating human GDF15 concentrations (~4ng/ml or more) are likely dependent on tumor-derived GDF15 for cachexia induction, we next sought to determine if intermediate serum concentrations of human GDF15 (~2ng/ml) were sufficient to support cachexia-associated adipose and muscle wasting induced by NSCLCs possessing STK11/LKB1 loss of function variants. The parental human 1944 NSCLC line had average human GDF15 expression (see Fig. 1B) and intermediate serum concentrations (~2ng/ml; see Fig. 1D) compared to the other STK11/LKB1 mutant CC inducing lines. We therefore separately silenced GDF15 (H1944\DeltaGDF15) or GFP (H1944\DeltaGFP) in the parental H1944 cells using lentiviral CRISPR/Cas9 techniques. Immunoblot analysis verified that that the monoclonal H1944\DeltaGDF15 line had silenced GDF15 protein expression compared to parental H1944 and H1944\DeltaGFP control cell lines (Supplementary Fig. S5A). These parental and engineered cell lines were subsequently injected subcutaneously into NOD/SCID immunodeficient mice and tumors derived from the H1944\DeltaGDF15 cells showed persistent elimination of GDF15 protein expression by immunoblot analysis (Supplementary Fig. S5A). There was no significant change in tumor growth when comparing the parental H1944, H1944\DeltaGFP, and H1944\DeltaGDF15 tumors (Supplementary Fig. S5B). Serum from mice harboring H1944\DeltaGDF15 tumors had a significant decrease in human (tumor cell-derived) GDF15 concentration compared to the serum of mice injected with parental H1944 or H1944\DeltaGFP cells (Supplementary Fig. S5C). However, serum mouse (host cell-derived) GDF15 concentrations remained low in all three cohorts (Supplementary Fig. S5D). In this model, mice harboring H1944\DeltaGDF15 tumors had a significant increase in their daily food intake (Supplementary Fig. S5E) with a preservation of tumor-free body weight (Supplementary Fig. S5F), tumor-free lean mass (Supplementary Fig. S5G), and fat mass (Supplementary Fig. S5H). These studies confirmed that intermediate concentrations of circulating GDF15 derived from an STK11/LKB1-mutated NSCLC line can also induce the cachexia phenotype, with the cachexia rescued by functional genetic targeting of tumor cell GDF15 expression.

Using GDF15 neutralizing antibody and/or tumor cell GDF15 silencing studies, we were able to clearly show that cancer cachexia models driven by STK11/LKB1 loss of function tumor variants with high and intermediate concentrations of circulating GDF15 were dependent on this tumor-derived factor to promote cachexia-associated fat and muscle atrophy. We used the next set of studies to establish a lower GDF15 concentration boundary of dependence by STK11/LKB1-mutated NSCLC lines for promoting cachexia wasting. Although immunodeficient mice harboring STK11/LKB1-mutated H2122 tumors induce cachexia when injected subcutaneously into NOD/SCID mice, their human (tumor-derived) GDF15 mRNA expression (see Fig. 1B) and GDF15 serum concentrations (see Fig. 1D) were at levels similar to mice bearing NSCLCs with wild-type STK11/LKB1 that could not induce cachexia. Next, we silenced GDF15 (H2122\DeltaGDF15) or GFP (H2122\DeltaGFP) in the parental H2122 cells using lentiviral CRISPR/Cas9 techniques. Immunoblot analysis revealed that the H2122\DeltaGDF15 line had suppression of GDF15 protein expression compared to parental H2122 and H2122\DeltaGFP cell lines (Fig. 6A). These cell lines were subsequently injected subcutaneously into NOD/SCID immunodeficient mice followed by measurement of the wasting phenotype. There was no significant difference in tumor growth among the three tumor cohorts (Fig. 6B) despite the persistent suppression of GDF15 protein expression from the H2122\DeltaGDF15 tumors (Fig. 6A). Tumor weight measurements conducted at study end showed H2122\DeltaGDF15 tumor weights were similar to tumor weights compared to H2122 parental tumors, but significantly decreased compared to H2122\DeltaGFP tumors (Fig. 6C). Mice harboring the parental H2122 or the H2122\DeltaGFP lines had average serum human (tumor cell-derived) GDF15 concentrations less than 0.5ng/ml (Fig. 6D). Although this was significantly increased compared to PBS-treated mice, this concentration of GDF15 was an order of magnitude lower than observed in the H1573 (see Fig. 2D and Supplementary Fig. S2C) mouse studies. As expected, mice harboring H2122\DeltaGDF15 tumors had a significant decrease in human (tumor cell-derived) GDF15 concentrations (Fig. 6D). Mouse GDF15 ELISAs showed no significant serum expression of GDF15 in the models tested (Fig. 6E). With respect to resulting phenotypes, we did observe a significant increase in food intake in the mice harboring H2122\DeltaGDF15 tumors compared to those mice bearing parental tumors (Fig. 6F). There was also a significant preservation of tumor-free body weight (Fig. 6G), tumor-free lean mass (Fig. 6H), and fat mass (Fig. 6K) in the experimental cohorts compared to the parental H2122- and H2122\DeltaGFP-bearing mice. More specifically, staining of muscle tissue (Fig. 6I) demonstrated larger myocyte cross-sectional area (Fig. 6J) in mice bearing GDF15-silenced tumors than in control tumor bearing cohorts. Similarly, H&E staining of adipose tissue (Fig. 6L) showed larger adipocyte cross-sectional area (Fig. 6M) and a distribution of areas shifted towards larger sizes (Fig. 6N) in mice bearing GDF15-silenced tumors. Overall, tumor-secreted GDF15 also contributes to the cachexia phenotype in mice bearing STK11/LKB1-mutated NSCLC lines demonstrating low systemic concentrations of the protein.

Dependence of STK11/LKB1-mutated NSCLC cachexia on GDF15 is verified in syngeneic models
Previously, all experiments interrogating cachexia biology in the context of STK11/LKB1 tumor loss and GDF15 relevance had prioritized using human NSCLCs derived from patients in immunodeficient murine models. To determine if similar findings could be recapitulated with an intact immune system, we obtained murine isogenic tumors cell lines derived from well-established KP and KPL genetically-engineered mouse models (GEMMs)31,32. The KP model has mutations in Trp53 and Kras leading to functional inactivation of both genes whereas the KPL model has an additional inactivating mutation in Stk11/Lkb1. Use of tumor cells derived from these murine lines in allotransplant experiments permitted us the ability to titrate the timing of tumor generation and kinetics of tumor growth in the context of a fully functional immune system. As expected, the KPL cell line expressed no STK11/LKB1 and had limited phosphorylation of AMPK (Supplementary Fig. S6A) compared to the KP line determined by immunoblot analysis. KPL tumors had a trend of increased growth kinetics (Supplementary Fig. S6B) and significantly increased tumor weights (Supplementary Fig. S6C) compared to KP tumors, a result observed by others and our previous work when silencing STK11/LKB1 in tumors transplanted into syngeneic models1,31. Food intake was reduced in the mice bearing KPL tumors compared to the control cohorts (Supplementary Fig. S6D). In Supplementary Fig. S6E, KPL-bearing mice demonstrated elevations in serum GDF15 concentrations above baseline/PBS-injected control animals. Although there was ~2-fold increase in serum mouse GDF15 levels in KPL-bearing mice compared to KP-bearing mice, these serum concentrations were lower than all of the patient-derived STK11/LKB1-mutated NSCLC lines previously tested (see Fig. 1D). Unlike tumors formed from the KP line, however, those formed from transplantation of the KPL line were able to induce host cachexia-associated tumor-free body weight loss (Supplementary Fig. S6F), tumor-free lean mass loss (Supplementary Fig. S6G), and fat loss (Supplementary Fig. S6H) in the mice into which they were transplanted.

To determine if GDF15 is relevant to cachexia induced in KPL-bearing animals, we suppressed GDF15 action with the administration of a GDF15 neutralizing antibody to specific KPL-bearing mouse cohorts. PBS and non-specific IgG were administered to the control KPL cohorts. These studies verified that KPL-induced wasting metrics – anorexia (Fig. 7C), tumor-free body weight loss (Fig. 7D), fat loss (Fig. 7E), and lean mass loss (Fig. 7H) were dependent on GDF15 despite similar tumor growth (Fig. 7A) and terminal tumor weights (Fig. 7B). Finally, we verified that the GDF15 antibody was able to suppress KPL-mediated, cachexia-associated decreased adipocyte (Fig. 7F, G) and muscle (Fig. 7I, J) cross-sectional areas. Overall, this syngeneic KP/KPL model further validated GDF15’s role in STK11/LKB1-mutated cachexia induction even at low serum concentrations.

Reconstitution of wild-type STK11/LKB1 verifies its role in regulating cachexia through regulation of GDF15 expression and release
To assess if we could abrogate cachexia induction in STK11/LKB1-mutant NSCLC lines with reconstitution of wild-type STK11/LKB1 protein, we identified the STK11/LKB1-mutated H1437 line that does not make endogenous STK11/LKB1 at baseline due to a deletion of exons 2 and 31. Tumor-bearing parental H1437 mice had the second highest tumor mRNA expression and the highest serum concentrations of human tumor-cell-derived GDF15 when compared to the other STK11/LKB1-mutant lines and the non-cachexia lines as well (see Fig. 1B, D). Again, we established the importance of GDF15 to this STK11/LKB1-mutated NSCLC in vivo by silencing the expression of the cytokine with CRISPR/Cas9 techniques (Supplementary Fig. S7A), leading to the elimination of the protein in its precursor or mature form in cells or medium. Injection of mice with H1437\Delta1437\DeltaGDF15 cells had no effect on tumor volume (Supplementary Fig. S7B), tumor weight (Supplementary Fig. S7C), or increase in food intake (Supplementary Fig. S7F) even though there was suppression of human serum GDF15 levels (Supplementary Fig. S7D) with no effect on murine serum GDF15 levels (Supplementary Fig. S7E). H1437\DeltaGDF15 tumor bearing mice were protected against body weight loss (Supplementary Fig. S7G), lean mass loss (Supplementary Fig. S7H), and fat mass loss (Supplementary Fig. S7I). GDF15 silencing also prevented reductions in myocyte (Supplementary Fig. S7K) and adipocyte cross-sectional areas (Supplementary Fig. S7L) compared to controls, measured from relevant muscle and adipose tissue staining, respectively (Supplementary Fig. S7J). The H1437\DeltaGFP tumor-bearing mice had a significant decrease in systemic leptin concentrations compared to the H1437\DeltaGDF15 tumor-bearing mice (Supplementary Fig. S8C), once again providing a possible explanation for the lack of change in food intake in this model.

We next overexpressed the wild-type gene using a lentivirus system and made stable H1437 STK11 tumor lines. We also created a monoclonal control line that received only empty vector (H1437CTL). As observed from immunoblotting analysis in Fig. 8A, we were successfully able to create two monoclonal lines overexpressing wild-type STK11/LKB1 (H1437STK11), associated with the expected increase in phosphorylation of AMPK compared to the parental H1437 and the H1437CTL tumor lines. This finding was verified in the stable H1437STK11 lines created in addition to the tumors derived from these lines in vivo. We next tested these cells in conditions of nutrient rich (high glucose) and nutrient deprived (low glucose) in vitro medium conditions, especially knowing that GDF15 mediates nutrient-limited states through the activation of integrated stress response (ISR) proteins CHOP and ATF413. The endoplasmic reticulum ISR signaling pathway includes multiple molecules, including PERK activation and its phosphorylation of eIF2\alpha, and subsequent nuclear ATF4 translocation supporting its transcriptional activity13. In the parental 1437 and H1437CTL NSCLC lines devoid of wild-type STK11/LKB1 expression, several gene products, including GDF15 and the ISR proteins CHOP and ATF4, were increasingly induced with decreasing glucose/nutrients (Fig. 8B). However, when wild-type STK11/LKB1 was reconstituted into the parental 1437 NSCLC line, decreasing medium glucose did not elicit an exaggerated ISR as observed by decreased ATF4 through immunoblot analysis and was associated with decreased cellular and secreted GDF15 (pro and mature forms). CHOP expression was relatively unchanged when comparing lines with wild-type and mutant STK11/LKB1 protein. Tumors derived from transplantation of the 1437 line reconstituted with wild-type STK11/LKB1 maintained expression of the kinase while still suppressing expression of all forms of GDF15. The tumor ISR signaling pathway was reduced as shown by a reduction in PERK levels and phosphorylation of the translation initiation factor eIF2 as expected (Fig. 8C). This data provided evidence of a clear mechanistic interaction between the presence or absence of STK11/LKB1 protein and the induction of the ISR with subsequent expression and processing of GDF15, the latter necessary for cachexia development in vivo. As expected, overexpression of the wild-type version of the tumor suppressor STK11/LKB1 led to a moderate suppression of tumor growth kinetics compared to control tumors (Fig. 8D), but animals were only sacrificed for cachexia evaluations once all tumors from all cohorts reached similar terminal sizes (Fig. 8D).

In parallel to the cell and tumor findings, mice bearing the STK11/LKB1-reconstituted H1437STK11 tumors demonstrated human serum GDF15 concentrations that approached baseline levels (i.e. equivalent to mice bearing no tumor) in comparison to the elevated serum GDF15 concentrations observed in mice bearing parental 1437 tumors or 1437 tumors overexpressing control vectors (Fig. 8E). The decrease in serum human GDF15 was associated with an increase in food intake in H1437STK11 tumor bearing mice compared to H1437CTL tumor bearing mice to levels similar to PBS-injected mice (Fig. 8F). Mice bearing H1437STK11 tumors displayed a preservation of body weight (Fig. 8G), fat mass (Fig. 8H), and lean mass (Fig. 8I) when compared to mice bearing parental 1437 or 1437ctrl tumors. Quantitative RT-PCR analysis of the cell lines demonstrated that the reconstitution of STK11/LKB1 profoundly limited the expression of the ISR-related molecules GDF15 and ATF4 compared to expression in control cells (Fig. 8J).

Since reconstitution of wild-type STK11/LKB1 decreased transcription and protein expression of GDF15, we could not assess the role of STK11/LKB1 in inducing the processing of the proprotein form of GDF15. To clarify this question, we overexpressed the FLAG-tagged version of the pro form of GDF15 into H1437CTL and H1437STK11 cell lines (Fig. 8K). Immunoblot analysis using anti-FLAG antibody revealed that the lack of STK11/LKB1 allowed for processing of the proprotein form of GDF15 into the mature protein in cells that was secreted into the medium. Whereas, overexpression of this construct into the wild-type STK11/LKB1 reconstituted cells only showed the proprotein form of GDF15 in the cells without the processed/mature form found in the cells or secreted into the medium. Furthermore, mature GDF15 protein is similarly lost in this system (medium and cells) with silencing of ATF4 in H1437 cells (Fig. 8L). The effect on eliminating mature GDF15 levels in cells or medium was not correlated with any change in GDF15 RNA expression with the absence of ATF4 protein (Fig. 8M). Ultimately, findings from these experiments continue to highlight a dependence of STK11/LKB1-mutated NSCLC tumors on GDF15 protein expression and processing for the induction of cachexia wasting. Reciprocally, the studies highlight the role of the stress response molecules STK11/LKB1 and ATF4 in promoting GDF15 processing into a mature, active form of the protein.

Methods
Study approval
Animal studies were approved through the University of Texas Southwestern Medical Center’s Institutional Animal Care and Use Committee (protocol 2015-100994).

Materials
Detailed material information is listed in Supplementary Table 1.

Buffer and culture medium
Buffer A contained 10 mM Tris-HCl (pH 6.8), 100 mM NaCl, 1% (w/v) SDS, 1 mM EDTA, 1 mM EGTA, Phosphatase Inhibitor Cocktail Set I and Set II, and Protease Inhibitor Cocktail. Medium A was RPMI 1640 supplemented with 5% (v/v) fetal bovine serum (FBS), 100 units/ml penicillin, and 100 \mu g/ml of streptomycin. Medium B was DMEM high glucose supplemented with 100 units/ml penicillin and 100 \mu g/ml of streptomycin. Medium C was medium B supplemented with 10% (v/v) FBS. Medium D was RPMI 1640 without glucose supplemented with 0.5% FBS, 100 units/ml penicillin, and 100 \mu g/ml of streptomycin.

Cell culture
Stock cultures of all human NSCLC cell lines (kind gift from John Minna), KP, KPL cell line (kind gift from Esra Akbay), and HEK293T cells (ATCC) were maintained in monolayer culture at 37 °C in 5% CO2 in medium A or medium C, respectively. Each cell line was propagated, aliquoted, and stored under liquid nitrogen. Aliquots of these cell lines were passaged for less than 4 weeks to minimize genomic instability prior to injection into mice. Every six months, the cell lines were tested for Mycoplasma contamination using the MycoStrip Mycoplasma Detection Kit.

Generation of gene knockout CRISPR/Cas9 lentivirus
A single guide RNA (sgRNA) directed to GFP as non-targeting control (\DeltaGFP) or to human GDF15 (\DeltaGDF15) were cloned into the BsmBI (Thermo, #ER0451) site of lentiCRISPRv2-puro vector (AddGene, #52961) using the following pairs of annealed oligonucleotides obtained from IDT for GFP (forward 5′-CACCGGTGAACCGCATCGAGCTGA-3′; reverse 5′-AAACTCAGCTCGATGCGGTTCACC-3′), or human GDF15 (forward 5′-CACCGGGACGTGACACGACCGCTG-3′; reverse 5′-AAACCAGCGGTCGTGTCACGTCCC-3′), or human ATF4 (forward 5′-CACCGATCCACAGCCAGCCATTCGG-3′; reverse 5′-AAACCGAATGGCTGGCTGTGGATC-3′). These constructs were transformed into stable competent E. Coli (NEB, #C3040H) and constructs were purified using the manufacture’s instruction of the MIDI-Prep system (Macherey-Nagel, #740410.10). The cloned nucleotides were confirmed by sanger sequencing. For lentiviral production, HEK293T cells were seeded in 100 mm dishes at ~1 \times 10^6 cells in 10 ml medium D on day 0. On day 2 (~70–80% confluence), medium was then removed and replaced with 8 ml medium C supplemented with an additional 2 mM glutamine, and 15 \mu l X-tremeGENE HP DNA Transfection Reagent, 2.5 \mu g of the indicated sgRNA-lentiCRISPRv2, 1 \mu g of pMD2.G (AddGene, #12259), and 1.5 \mu g of psPAX2 (AddGene, #12260) mixed in 0.8 ml Opti-MEM medium (Thermo, #31985070). On day 3, medium was removed and treated with 11 ml medium B with 30% FBS supplemented with an additional 2 mM glutamine. On day 4, 5, and 6, virus-containing medium was collected and exchanged with 11 ml of fresh medium B with 30% FBS supplemented with an additional 2 mM glutamine. The collected virus-containing medium was filtered through a 0.45 \mu m syringe filter unit (Millipore Sigma) and stored at −80 °C for future use.

Generation of wild-type STK11/LKB1 and GDF15-3x-flag overexpression lentivirus
Complimentary DNA library was derived from the reverse transcription (Thermo, #N8080234) of extracted RNA from the H1792 line as described by the manufacturer’s directions. Human STK11 (NM_000455.5) complimentary DNA containing the EcoRI and BamHI restriction sites at the 5′ and 3′ ends, respectively, was amplified using the following pair of primers (forward 5′-TATTTCCGGTGAATTCatggaggtggtggacc-3′; reverse 5′-GAGAGGGGCGGGATCCtcactgctgcttgcag-3′). This fragment was cloned into the pLVX-EF1\alpha-IRES-Puro vector (Clontech, #631988) by first treating with the EcoR1-HF (NEB, #R3101S) and BamH1-HF (NEB, #R3136S) restriction enzymes followed by In-Fusion Snap Assembly (Takara, #638947) per the manufactures’ directions. Human GDF15 (NM_004864.2) ORF was purchased from UT Southwestern McDermott Center for Human Genetics, that was a clone from the Ulitmate ORF Lite human cDNA collection (Life Technologies). GDF15-3x-Flag complimentary DNA containing the XbaI and BamHI restriction sites at the 5′ and 3′ ends, respectively, was amplified using the following pairs of primers (forward 5′- CGAGACTAGTTC TAGAGCCACCatgcccgggcaagaac-3′; reverse 5′- GAGAGGGGCGGGATCCctacttgtcatcgtcatccttgtaatcgatatcatgatctttataatcaccgtcatggtctttgtagtctatgcagtggcagtc-3′). This fragment was cloned into the pLVX-EF1\alpha-IRES-Hygro vector (kindly gifted by Shimeng Xu, UTSW Medical Center) by first treating with the XbaI (NEB, #R0145S) and BamH1-HF (NEB, #R3136S) restriction enzymes followed by In-Fusion Snap Assembly (Takara, #638947) per the manufactures’ directions. These constructs were transformed into stable competent E. Coli (NEB, #C3040H) and constructs were purified using the manufacture’s instruction of the MIDI-Prep system (Macherey-Nagel, #740410.10). The cloned nucleotides were confirmed by sanger sequencing. For lentiviral production, HEK293T cells were seeded in 100 mm dishes at ~1 \times 10^6 cells in 10 ml medium D on day 0. On day 2 (~70–80% confluence), medium was then removed and replaced with 8 ml medium C supplemented with 2 mM glutamine, and 15 \mu l X-tremeGENE HP DNA Transfection Reagent, 2.5 \mu g of the indicated pLVX-EF1\alpha-IRES-Puro construct, 1 \mu g of pMD2.G (AddGene, #12259), and 1.5 \mu g of psPAX2 (AddGene, #12260) mixed in 0.8 ml Opti-MEM medium (Thermo, #31985070). On day 3, medium was removed and treated with 11 ml medium B with 30% FBS supplemented with an additional 2 mM glutamine. On day 4, 5, and 6, virus-containing medium was collected and exchanged with 11 ml of fresh medium B with 30% FBS supplemented with an additional 2 mM glutamine. The collected virus-containing medium was filtered through a 0.45 \mu m syringe filter unit (Millipore Sigma) and stored at −80 °C for future use.

Generation of knockout or overexpression cell lines
Monoclonal parental, empty vector overexpressed, or wild-type STK11/LKB1 overexpressed H1437 NSCLC lines were seeded in 6-well dishes at ~1 \times 10^5 cell density in 2 ml of medium A. After 24 h the medium was replaced with fresh medium A (non-transduced control) or 1 ml of the indicated virus-containing medium, supplemented with 8 \mu g/ml polybrene reagent (Millipore Sigma, #TR-1003). After 24 h, the medium was removed and replaced with 2 ml of medium A. The next day, the medium was removed and replaced with fresh 2 ml medium A containing 1 \mu g/ml puromycin or 300 \mu g/ml hygromycin, and puromycin- or hygromycin-supplemented medium A was refreshed every 2 days until complete cell death was observed in the non-transduced control. For knockout or wide-type STK11/LKB1 overexpression, limiting dilution of these puromycin-selected cells was performed in puromycin-containing medium to produce single colonies of transduced cells, and these monoclonal knockout lines were subsequently propagated and aliquoted for storage under liquid nitrogen or further propagated for experiments described herein.

Cancer cachexia animal models
All animal studies were conducted under an Institutional Animal Care and Use Committee (IACUC)-approved protocol at UT Southwestern Medical Center (Dallas, Texas). NOD.CB17-Prkdc<scid>/J (NOD/SCID) and C57BL/6J mice were obtained from The Jackson Laboratory at approximately 9 weeks of age. All mice were allowed to acclimate in UT Southwestern animal facilities for at least 2 weeks. Animals were kept in a temperature-controlled facility (at ~22 °C) with a 12 h light/dark cycle and were fed regular chow diet.

On day 0 of the study, NOD/SCID or C57BL/6J mice underwent baseline assessments of body weight (digital Ohaus scale) and both lean and fat mass components of body composition using ECHOMRI (ECHO Medical Systems).

For subcutaneous model, 200 \mu l PBS in the absence or presence of either 1 \times 10^7 human or 1 \times 10^6 mouse NSCLC cells (parental or genetically engineered lines) were injected into the right flank of the NOD/SCID or C57BL/6J mice, respectively.

For orthotopic model, the procedure for surgical orthotopic transplantation of lung cancer cells has been published previously27–29. Briefly, a 1-cm incision was made through the skin and underlying subcutaneous fat of mice anesthetized under continuously metered 2% isoflurane. PBS or 1 \times 10^6 tumor cells (H1573 GFP-KO or GDF15-KO) suspended in 30 \mu l of 25% growth factor reduced Matrigel in dye-free RPMI was injected via a 30-gauge needle through the intercostal muscles directly into left lung, respectively. Skin was stapled together using 9-mm staples. Carboprofen 5 mg/kg was administered intraperitoneally after surgery and the following two days for pain control. Mice were monitored until fully recovered from anesthesia. After 12 days’ recovery, staples were removed, and mice were monitored as below.

Food intake measurements were conducted daily as described previously, including the method for calculation of average daily food intake25. Every 2–4 days, mice underwent longitudinal measurements of body weight, both lean and fat mass components of body composition, and tumor size by caliper (VWR) measurements of length, width, and breadth.

UT Southwestern IACUC policy for single subcutaneous tumor requires euthanasia of animals when the diameter of any one tumor is greater than 2 cm in a mouse. The timing of animal sacrifice for tumor-injected cohorts was a function of imminent animal death (expected within 12 h), tumor approaching 2.0 cm diameter, or cohorts of animal reaching about 50% fat loss; PBS cohorts were sacrificed concurrently with matched tumor cohorts. At sacrifice, mice were euthanized as recommended by the IACUC with use of a CO2 chamber. Tumor weights were measured and collected and snap frozen in liquid nitrogen and placed in long-term storage at −80 °C (for future analysis). Whole blood at sacrifice was obtained through cardiac puncture and serum was obtained by subjecting the whole blood to centrifugation at 960 \times g at 4 °C for 10 min followed by collection of the supernatant.

Longitudinal values for tumor volume were calculated as half the product of caliper measurements of length, width, and breadth. Longitudinal and terminal values for body weight, lean mass, and fat mass were calculated relative to both the matched animal day zero value and mean value of the matched PBS cohort. At sacrifice, relative tumor-free body weight and lean mass were calculated by subtracting the matched animal tumor weight at sacrifice prior to normalization.

Serum protein concentration determination
Human GDF15 in serum was measured using a human GDF15 Quantikine ELISA kit (R&D Systems, #DGD150,) following the manufacturer’s instructions. Mouse GDF15 in serum was measured using a mouse/rat GDF15 Quantikine ELISA kit (R&D Systems, #MGD150) following the manufacturer’s instructions. Mouse Leptin in serum was measured using a mouse ELISA kit (CrystalChem, #90030) following the manufacturer’s instructions.

Immunoblot analysis
Human NSCLC cell lines cells were seeded in 6-well dishes at ~4 \times 10^5 cell density in 2 ml of medium A. After 24 h cells were washed with 2 ml DPBS, then treated with Medium A or Medium D in the absence or presence of different concentrations of glucose as indicated. After 16 h, media were harvested and spun with 800 \times g for 2 min and the supernatant was concentrated 15-fold using a 3kDa filter (Amicon Ultra) per manufacturer’s instructions to obtain the concentrate. Cells were washed with 2 ml DPBS, then cells were treated with 170 \mu l of buffer A then subjected to a plate shaker for 2 min at 700 RPM followed by harvesting of the cell lysate which was subjected to 10 s of sonication (Cole-Parmer, #EW-04714-50) at 50% amplitude. The concentrated media and cell lysate were then used for immunoblot analysis.

Tumor from mouse studies were thawed on ice and ~50–60 mg of tumor tissue was placed in a 2 ml microtubes with 1 ml of Buffer A and homogenized with a Bead Ruptor Elite apparatus (Omni International) for three cycles of 20 s at 4 m/s, followed by 10 s of sonication (Cole-Parmer, #EW-04714-50) at 50% amplitude. The homogenized sample was immediately subjected to centrifugation at 10 °C for 10 min at 20,000 \times g. The supernatant was separated from the pellet and collected. The tissue lysate were then used for immunoblot analysis.

Protein concentrations of cell lysate, or mouse tumor lysate were measured using a PierceTM bicinchoninic acid kit and then mixed with 5X loading buffer (250 mM Tris-HCl (pH 6.8), 10% sodium dodecyl sulfate, 25% glycerol, 5% \beta-mercaptoethanol, and 0.2% bromophenol blue) heated at 95 °C for 5 min, and subjected to 10% or 15% SDS/PAGE (8 \mu g/lane for cell/tumor lysate, or volume of cell medium normalized to cell protein concentration). The electrophoresed proteins were transferred to nitrocellulose membranes using the Bio-Rad Trans Blot Turbo system, followed by incubation in blocking buffer containing 5% (w/v) non-fat powdered milk in PBS-T. Blocked membranes were washed briefly in PBS-T, followed by primary staining with indicated primary antibodies diluted in primary antibody solution-Can Get Signal (TOYOBO). Membranes were again washed in PBS-T, followed by secondary staining with donkey anti-mouse or rabbit IgG conjugated to horseradish peroxidase diluted in the blocking buffer. Membranes were again washed in PBS-T, and bound antibodies were visualized by brief incubation in chemiluminescent substrate solution followed by imaging using an Odyssey FC Imager Dual-mode Imaging System (2-min integration time). Immunoblot images were analyzed using Image Studio software (LICOR).

Real-time PCR analysis of gene expression.
Extraction of total RNA with RNA-STAT-60 reagent from cells, tumors, and muscles, subsequent quantitative reverse transcriptase PCR assays for mRNA levels of the indicated gene products were conducted as previously described (1). The sequences of primers (obtained from Integrated DNA Technologies) used in these studies are as follows:

Gene product Symbol Forward Reverse
\beta-actin ACTB CACCATTGGCAATGAGCGGTTC AGGTCTTTGCGGATGTCCACGT
Growth differentiation factor 15 (GDF15) Gdf15 (Mouse) CTCTCAACTGAGGTTCCTGC TCTGGACTGCCAATCTCACC
F-box protein 32 (Fbxo32) Fbxo32 GAGGGCCATTGACTTTGGGAC CTCCTTCTTCATGTGTTCTTCT
Tripartite motif containing 63 (Trim63) Trim63 GAGGGCCATTGACTTTGGGAC CTCCTTCTTCATGTGTTCTTCT
Activating transcription factor 4 (ATF4) ATF4 CTCCAACAACAGCAAGGAGGA TACCCAACAGGCATCCAAG
Sarcolipin Sln TGTGCCCCTGCTCCTCTTC TGATTGCACACCAAGGCTTG

Microscopic analysis of mouse adipose, muscle tissue and lung tumor area.
Epididymal white fat was collected, and immersion fixed in 10% neutral-buffered formalin. All samples were paraffin processed by members of UT Southwestern’s Histo Pathology Core using standard histologic technique. Resulting embeds were sectioned at 5 \mu m, and produced slides were stained by regressive methodology utilizing Leica Selectech reagents (Hematoxylin 560, Define, Blue Buffer, and Eosin-Y 515) on a Tissue-Tek Prisma Plus robotic stainer (Sakura-USA). From the stained slides, microscopy fields were imaged on a BZ-X scanner (Keyence) at 20\times magnification and adipocyte area was measured using ImageJ software Adiposoft plugin (Fiji) for three fields from each slide (250–350 adipocytes per slide). Each group contains the quantification of slides prepared from indicated number of mice.

Cryosections of whole Gastrocnemius muscle were prepared by UT Southwestern’s Histo Pathology Core and preserved at −80 °C freezer. When staining, slides were brought to room temperature and surrounded with liquid blocker. Slides were blocked with blocking buffer (5% goat serum in PBS) in a wet chamber for at least 30 min at room temperature. Slides were incubated with Laminin antibody (1:1000 in blocking buffer) overnight at 4 °C. The next day, slides were washed with PBS 3 times. Slides were incubated for at least 1 h with secondary antibody (Anti-rabbit-Alexa Fluor 488 was diluted 1:1000 in blocking buffer). Slides were washed with PBS 4 times. Slides are covered with immunostaining mounting media and cover slides. Fluorescent images were captured using the Keyence BZ-X700 microscope. Three fields from each slide were imaged and myofiber cross-sectional areas were measured using Ilastik (EMBL Heidelberg) and ImageJ (Fiji) software34.

Paraffin embedded blocks of lungs were made in UTSW Tissue Management Shared Resource Facility. H&E images of left lungs were scanned with Hamamatsu NanoZoomer 2.0 HT in UTSW Whole Brain Microscopy Facility. Images visualization, tumor and lung areas were calculated using Hamamatsu NDP View 2 software. Analysis of the percentage of tumor area was performed using GraphPad Prism 10.

Forelimb grip strength test
Grip strength was measured using a BIO-GS4 grip strength test meter (Bioseb Instruments, France). Mice were placed on a metal grid connected to the meter and slowly pulled by the tail. The meter records the maximum grip strength (gram, and was converted to newton (N) by multiplying 0.0098). The measurement was repeated five times and averaged to determine the grip strength of each mouse. Relative grip strength is normalized with day 0 body weight and shown in Supplementary Data 1.

Statistics & reproducibility
For subcutaneous models, each group includes 6 mice; for orthotopic model, each group includes 9 mice based on previously published literature. Mice data points were excluded from the results when there were no tumor forming or mice died unexpected before data collection. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment.

Details of statistical analysis for each experiment can be found in the respective figure legend. Data is presented as mean \pm SEM, dot plots \pm SEM, dot plots with bars \pm SEM, or histogram. For experimental designs with two conditions, unpaired parametric t tests were conducted to investigate significant differences following tests to verify normality and equal variance of both groups. For experimental designs with greater than two conditions, one- or two-way analysis of variance (ANOVA) tests were conducted to examine if there were significant differences in outcomes among groups. For the case of multiple pairwise comparisons, Dunnett’s multiple comparison post-test was used. All statistical analyses were computed using GraphPad Prism 10 software.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The current manuscript describes the impact of pharmacologic and genetic GDF15 targeting to prevent lung cancer (NSCLC)-induced cachexia. The importance of GDF15 in cancer cachexia was previously reported and anti-GDF15 clinical trials in NSCLC patient are currently ongoing. The main novelty of the manuscript looks the GDF15 secretion to depend from LKB1 mutations independently from anti-cancer therapies and that the mutations in the tumor may predict GDF15 production and cachexia development. The association between GDF15 production and LKB1 activity was previously reported (PMID: 38111379) in non-cancerous cells and several studies highlighted the link between GDF15 and AMPK. However, the mechanism linking the AMPK/LKB1 pathway modulation with GDF15 secretion is missing from both previous and current study. The research was mainly conducted implementing the use of subcutaneous xenotransplantation of human cells in immunodeficient mice, producing a limited translatable condition to human cachexia (acknowledged in the discussion). The authors thus implement the use of iso-/syn-graft in immunocompetent mice, obtaining non completely overlapping, although coherent data. Oveall, the results are interesting and deserve attention, however, there are several points that require a careful revision either by providing new / newly elaborated data or by argumenting.\nMain issues:\nThe introduction is in my opinion too long and resembles partly a result and partly a discussion chapter, failing to provide the state of the art in the studied field in order to understand the following results. Considering that cancer cachexia does not develop in all cancer types, a brief description of the development of cancer cachexia in NSCLC could be added.\nAnimal sex is not specified. The authors should clarify which animal sex was chosen and explain the reason behind the choice.\nIn order to present homogeneous data for the different models adopted, the tumor and tissue weights at necropsy (at least muscle and adipose tissue) should be included for all the experiments. Considering that the lean mass assessment by ECHO-MRI cannot dissect the contribution of different tissues, and that the enlargement of a tissue could compensate for the reduction of another, the final tissue weights should be presented, even in a supplemental table.\nFigure 1: measuring host contribution to GDF15 production in immunodeficient mice is in my opinion inappropriate, since the host response to the presence of the cancer is severely impaired. The authors describe the association between CC presence and high tumor GDF15 transcript and released peptide, however a recent observational clinical trial suggest the lack of association between cachexia and high GDF15 (PMID: 37115357). Please discuss.\nFigure 2: tumor-free mice untreated, treated with IgG or anti-GDF15 are missing. Since the tumor-bearing mice treated with anti GDF15 gain weight/mass, I think it is important to show whether the anabolic response is present even in healthy controls despite having almost undetectable GDF15 levels.\nFigure 3: Daily food intake is not reduced in H1573 tumor bearing mice showing high GDF15 levels and GDF15 blockade has no impact on food intake. The role of GDF15 in cachexia has been mainly described as an anorexigenic molecule, while it is not the case in the current model. I think the authors should better describe the mechanism underlying GDF15 action in the currently adopted model. Surprisingly, when injecting H2122 cells in mice, anorexia is present and silencing GDF15 protects from anorexia, although H2122 cells produce less GDF15 than H1573 (Figure 5). Again, when using H1437 cells (Figure 7 and S5), it looks like that the reduction of GDF15 release obtained by wt LKB1 reconstitution allows to counteract anorexia. Such scattered results are in overt contrast with the authors conclusion stating that ‘Our studies further illustrated that when the GDF15 increases in serum concentrations were not as high, there was less effect on food intake’ (Line 477). Some values of daily food intake seem to be very high, going above 7g/day/mouse (such as in figure 3F). Can the authors explain such high values? Food intake kinetic per group could be an interesting addition. Have the authors checked for food grinding? How many mice were present in each cage? Was any mouse excluded, sacrificed before the established endpoint, producing a change in the feeding behavior of the remaining mice?\nFigure 4: adipocyte and muscle fiber CSA in healthy controls are missing. 3 mice per cohort for histology looks too low and unlikely to provide such statistical differences. I wonder whether the average CSA from each mouse has been used as a single point or each adipocyte/fiber area has been considered as an independent point. The second option is incorrect. In the third panel of Figure 4E, there is an unexpected increase in mice injected with ΔGFP cells; this highlights a possible limitation of having only 3 mice per cohort. The same applies for Figure 5H, where there is an unexpected reduced CSA in ΔGFP.\nFigure 6, panel B: is tumor volume at sacrifice statistically different? Tumor mass, anorexia, serum GDF15 and cachexia look directly correlated. In panel J form the same figure, it looks like that anti GDF15 initially promotes adipose mass accumulation in KPL-bearing mice, followed by a second phase of ‘resistance/ineffectiveness’. A similar phenomenon seems to occur in mice bearing H1792ΔSTK11 tumors (Supplemental figure 4, panel E). Given the potential translational value of the study, the prophylactic nature of the design is not fully convincing, suggesting to perform a therapeutic study to show the impact of anti GDF15 in early cachectic mice (i.e. starting at day 15 or 20 from KPL cell injection). Similarly, a survival (or even a ‘time to humane endpoint’) study would strongly increase the translational value. Panel K: first time showing skeletal muscle weight; I would recommend including healthy controls. Similarly, relative tumor-free body weight and relative tumor-free lean mass for KPL + IgG/GDF15ab are missing.\nFigure 7: very elegant gene reconstitution experiment, however, the reduced tumor growth rate in reconstituted LKB1 cells does not allow to conclude on the impact on cachexia, given that the same tumor mass developed in a different time frame likely induced a differential adaptation in the hosts, resulting in a different cachectic state. Panel D: the x axis of the graph has to be fixed.\nI hardly understand the supplemental figure 5, where two experiments almost identical show contrasting or even opposite results. How can 2 weeks of age difference in adult mice produce such different results?\nThe authors state that GDF15 is relevant for muscle contractility. Considering that in this work GDF15 signaling is blocked, an assessment of muscle functon(at least grip test and/or treadmill test, in case physiological muscle strength is not a lab commodity) should be included.\nMinor issues:\nIs it necessary to have 7 figures in the main text?\nFig4 legend, ‘H1573 NSCLC lines allotransplanted into murine models’: the cells are transplanted in mice, the model is obtained in the mouse transplanted with cells.\nWB: overexposed and low quality of the results.\nline 70, ‘that were are not’\nline 72, lack of isogenic models: the authors should clarify whether referring to the host, to the cancer cells, or both, given that there are several models of CC using syngeneic mice receiving almost monoclonal cancer cells originated from the same host animal strain.\nLine 87, ‘STK11/LKB1 functional variant’: do the authors refer to an oncogenic mutant? I suggest to briefly explain in the text.\nLine 146: please use the definition ‘cachexia-inducing cell line’ in spite of ‘cachexia line’.\nLine 221: despite having ECHO-MRI and CSA data, to state “adipose and muscle atrophy”, the final tissue weights should be reported.\nLine 237: missing comma after “food intake”.\nLine 482: “adipose and muscle tissues”.\nLines 483-485: the authors propose the use of metabolic cages and single-housing to better study food intake alterations associated with changes in GDF15 circulating levels. While single-housing can be an interesting approach to isolate the behavior/contribution of each animal, changing animal housing numerosity can be an independent factor affecting the feeding behavior, possibly determining discrepancies with the observations presented in this study. The authors should mention this limitation in the proposed future approach.\nLine 614: serum is obtained by collection, not removal of centrifuged whole blood supernatant.\nLine 643: why sonication at this point and not after the disruption?\nLine 859: the word “staining” is repeated.\nLine 889: missing a point at the end of the sentence.\nLine 927: “A-H”.\nLines 928, 944, 959: please adjust group numerosity reported in the text (doesn’t match with the related graphs).", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "This study from Yu et al. investigates the link between LKB1 loss and GDF15 in promoting cancer-associated cachexia in models of lung cancer. The authors demonstrate that loss of LKB1 in patient-derived lung tumor cells promotes an increase in GDF15 expression and a corresponding increase in circulating levels of GDF15. Treatment with an anti-GDF15 antibody alleviates features of cachexia in their cachexia inducing cell lines with mutation in LBK1/STK11. While the authors have demonstrated a clear link between loss of LKB1 and levels of GDF15 in their cancer cell lines, major reservations remain about the generalizability of this work due to the models of cachexia utilized in the manuscript. Additionally, there is little to no mechanistic insight provided into how loss of LKB1 regulates production of GDF15 both on the transcriptional and post transcriptional level. Major revisions are requested to this manuscript.\nMajor comments:\nThe study relies predominantly on subcutaneous tumor transplantation of a large number of cells, with tumors often reaching extremely large sizes (<1cm3) before any meaningful weight loss is observed. In human patients, tumor burden is often poorly predictive of weight loss associated with cachexia, with small tumors capable of inducing cachexia. A main concern over the validity of the conclusions of this manuscript is that these subcutaneous tumor models are poorly reflective of human disease, and do not accurately represent the complex interplay between tumor genetics and the tumor microenvironment. In an autochthonous tumor model of LKB1 deficient NSCLC, where tumors develop in the native environment of the lung, levels of GDF15 were shown to be elevated as compared to LKB1 WT tumors but play no part in phenotypes associated with cachexia. The link between LKB1 loss and GDF15, and the role of GDF15 in promoting cachexia in these tumors, should be studied in models of NSCLC where tumors grow in the native environment in the lung, including genetically engineered mouse models and syngeneic transplant studies. Such studies will give insight as to whether the link between GDF15 and LKB1 loss is robust or an artifact of the model chosen.\nThere seems to be an intrinsic effect on neutralizing GDF15 even in tumors that do not have high levels of circulating GDF15 (in particular, Supplemental Figure 4). This suggests that treatment with anti-GDF15 antibody is driving the reversal of cachexia phenotypes rather than through blocking the actions of GDF15. To confirm an on target effect of GDF15 neutralization, the anti-GDF15 antibody studies should be complemented with treatment of tumors with anti-GFRAL antibody, the only known receptor of GDF15. Additionally, previous studies have indicated that the principal contributor of GDF15 mediated weight loss is from sympathetic output rather than changes in food intake. It would be insightful to see the effect of chemical sympathectomy by 6-OHDA treatment to observe whether this output is playing a role in cachexia in these models.\nLKB1 regulates a host of downstream kinases including AMPK and the salt-inducible kinases (SIKs). Previous studies have indicated the tumor suppressive effects of LKB1 are dependent on the SIKs. Additionally, a CRTC2-CREB-SIK axis mediates the inflammatory potential of cells harboring loss of LKB1. It would be informative to see if increase in GDF15 expression is mediated through this same pathway or if other kinases downstream of LKB1 play a role.\nThe evidence linking lowering of GDF15 levels to STK11 reconstitution is clear in Figure 7, yet whether this lowering of GDF15 levels is primarily driving the improvement in weight loss is correlative. Studies utilizing GDF15 loss or neutralization should be performed to examine the role of the molecule in this context.\nMinor comments:\nGDF15 is a marker of metabolic stress – thus as tumors grow larger, it is expected that levels of GDF15 expression would increase as nutrient availability in the tumor diminishes and the metabolic demands for growth increase. Thus, it would be informative for the authors to provide relative tumor size effects on levels of GDF15 mRNA expression and circulating GDF15 levels.\nHow does relative bodyweight in tumor bearing animals change over time with GDF15 antibody administration? Only bodyweight at endpoint is provided.\nIt is unclear what is being blotted for in figure 3C, and the P and M labels are not described. It figure 3F, it seems there is both a loss of parental and mouse GDF15 levels in cells targeted with a guide against GDF15. In the food intake studies in figure 3F, while reaching statistical significance, the differences are extremely marginal and most likely not relevant to the relative weight loss.\nIn syngeneic experiments, what is the effect of GDF15 neutralization in the context of a KP tumor? This can provide more insight into the effect of the GDF15 antibody in the context of low levels of GDF15."}, "author_response": "Response to Referees\nDear Referees,\nWe thank you for your careful analysis of our paper and are very grateful for your\nconstructive comments. We have responded to the comments asked by the reviewers\nbelow, and we have incorporated appropriate changes into the manuscript. As a\nconsequence of addressing these comments through additional experimentation, we\nbelieve the quality of the manuscript has been strengthened significantly due to the\nincreased generalizability of our findings. Specifically, new Figure 4 is now the lung\northotopic xenotransplant model which correlates with our multiple subcutaneous models\nin the manuscript. Furthermore, new Figure 5 added GDF15 antibody rescue\nexperiments by initiating treatment after 20% fat mass loss. Suppressing cachexia\ndevelopment after it has already initiated increases translational therapeutic implications.\nAlso, in Figure 8, we offer additional mechanistic insight showing that STK1/LKB1-mutant\nNSCLC have an increased integrated stress response (ISR) that is critical to the\nproduction and processing of GDF15. Lastly, we find similar cachexia biology in both male\nand female mice. The details of these findings and our direct response to the reviewers’\ncritiques are found below.\nReviewer #1 Comments:\n1) “the mechanism linking the AMPK/LKB1 pathway modulation with GDF15\nsecretion is missing”: We now have generated additional data that offers significant insight\ninto the role that STK11/LKB1 plays when made functionally irrelevant in the setting of\nmore global stress sensing mechanisms. Our findings have highlighted that tumors with\nloss-of-function mutations in STK11/LKB1 have an exaggerated integrated stress\nresponse resulting in the production and processing of stress/cachexia-inducing cytokine\nGDF15. This activation occurs through an ER stress signaling pathway of PERK, peIF2α,\nand ATF4 (see Figures 8B-C). Not only does this increase in the integrated stress\nresponse cause elevated production of GDF15, it all appears that ATF4 induction also\nregulates the maturation of GDF15 from a pro to mature/secreted form (see Figures 8J-\nM). This promotion of GDF15 maturation is crucial since it leads to tumor cell specific\nrelease of the cytokine, which we have found to subsequently drive wasting. This\nmechanism becomes even more relevant since there does not appear to be an induction\nof or contribution from host tissues towards circulating GDF15 concentrations in our in\nvivo tumor cachexia models, prioritizing cell processing of the cytokine in specifically\nthose tumor cells with STK11/LKB1 alterations. Future efforts will explore the downstream\nsignaling mechanism and molecules critical to all aspects of GDF15 production and\nprocessing.\n2) “The introduction is in my opinion too long and resembles partly a result and partly\na discussion chapter, failing to provide the state of the art in the studied field in order to\nunderstand the following results. Considering that cancer cachexia does not develop in\nall cancer types, a brief description of the development of cancer cachexia in NSCLC\ncould be added.”: Introduction has been shortened and made more concise, all changes\nare now in red font.\n3) “Animal sex is not specified. The authors should clarify which animal sex as chosen\nand explain the reason behind the choice.”: As part of new experimentation included in\nSupplemental Figure 2, we verified no differences in cachexia development in vivo\nbetween male and female mice. GDF15-silenced malignancies showed similar tumor\nweights at experiment end and rescued all cachexia metrics including body weight, fat\nmass, muscle mass and grip strength in female mice (Supplemental Figure 2) similar to\nthose findings in male mice (Figures 2-3).\n4) “In order to present homogeneous data for the different models adopted, the tumor\nand tissue weights at necropsy (at least muscle and adipose tissue) should be included\nfor all the experiments. Considering that the lean mass assessment by ECHO-MRI cannot\ndissect the contribution of different tissues, and that the enlargement of a tissue could\ncompensate for the reduction of another, the final tissue weights should be presented,\neven in a supplemental table.”: Throughout all experiments, we have added additional\ntumor and cachexia-related metrics. As requested, we have now added tumor\nweights/areas in Figures 2C, 4B, 5C, 5J, 6C, 7B and Supplementary Figures 2B, 5C,\n6C.\na) As requested, we have also now added grip strength as a metric of muscle function\nin cachexia in Figures 2L and 4I and Supplemental Figure 2I.\nb) We have also added specific fat pad and muscle weights in Figures 3A, 3B, 3F,\n3G, 4J, 4M, 5N, 5P and Supplemental Figures 2J-M.\nc) From a histological perspective we have also evaluated adipocyte and myocyte\ncross sectional area in Figures 3C-E, 3H, 3I, 4K-L, 4N, 6I-J, 6L-N, 7F-G, 7I-J and\nSupplemental Figures 6J-L.\nd) In addition, we have also added muscle transcriptional changes that associate with\ncachexia/wasting in Figures 3J-K.\ne) To show that cachexia wasting was independent of tumor size, we also added\nFigure 1F comparing tumor sizes vs GDF15 serum levels in our patient-derived\nmodels.\n5) “Figure 1: measuring host contribution to GDF15 production in immunodeficient\nmice is in my opinion inappropriate, since the host response to the presence of the\ncancer is severely impaired.”: We have now added to our discussion the following\nstatement after we discuss that GDF15 is secreted from the tumor: “…understanding\nthe limitation of our immunodeficient model is missing part of the immune system.“\nAlthough we agree that immunodeficient models might inhibit the host response to\nincrease GDF15 under stress, most cell types that previously demonstrated GDF15\nproduction (macrophages- PMID: 9326641, granulocytes (PMID: 34381196), and\nmonocytes (PMID: 34381196) are still present in the NOD/SCID model which are\ndeficient in specifically T cell and B cell function.\n6) “The authors describe the association between CC presence and high tumor\nGDF15 transcript and released peptide, however a recent observational clinical trial\nsuggest the lack of association between cachexia and high GDF15 (PMID: 37115357).\nPlease discuss.”: The study mentioned by the reviewer employed an antibody directed\nagainst full length GDF15, whereas the GDF15 mature protein processed from a pro form\nis more likely to be released and is known to be the functional end-product identified in\ncirculation. This study also measured GDF15 levels in all comers, not just patients\npossessing STK11/LKB1-mutated tumors. Furthermore, the recently published Tracer-X\nstudy on cachexia (PMID: 37045997) showed an association between cachexia\ndevelopment and circulating mature GDF15 in recurrent NSCLC patients with cachexia.\n7) “Figure 2: tumor-free mice untreated, treated with IgG or anti-GDF15 are missing.\nSince the tumor-bearing mice treated with anti GDF15 gain weight/mass, I think it is\nimportant to show whether the anabolic response is present even in healthy controls\ndespite having almost undetectable GDF15 levels.” Tumor-free mice injected with PBS,\nIgG. or anti-GDF15 antibody were administered to NOD-SCID mice in new\nexperimentation and showed no cachexia phenotypic differences among cohorts. This is\npresented in new Supplemental Figure 3.\n8) “Figure 3: Daily food intake is not reduced in H1573 tumor bearing mice showing\nhigh GDF15 levels and GDF15 blockade has no impact on food intake. The role of GDF15\nin cachexia has been mainly described as an anorexigenic molecule, while it is not the\ncase in the current model. I think the authors should better describe the mechanism\nunderlying GDF15 action in the currently adopted model. Surprisingly, when injecting\nH2122 cells in mice, anorexia is present and silencing GDF15 protects from anorexia,\nalthough H2122 cells produce less GDF15 than H1573 (Figure 5). Again, when using\nH1437 cells (Figure 7 and S5), it looks like that the reduction of GDF15 release obtained\nby wt LKB1 reconstitution allows to counteract anorexia. Such scattered results are in\novert contrast with the authors conclusion stating that ‘Our studies further illustrated that\nwhen the GDF15 increases in serum concentrations were not as high, there was less\neffect on food intake’ (Line 477). Some values of daily food intake seem to be very high,\ngoing above 7g/day/mouse (such as in figure 3F). Can the authors explain such high\nvalues? Food intake kinetic per group could be an interesting addition. Have the authors\nchecked for food grinding? How many mice were present in each cage? Was any mouse\nexcluded, sacrificed before the established endpoint, producing a change in the feeding\nbehavior of the remaining mice?”: We do appreciate the earlier version inconsistencies\nin food intake among different models. But, with more experiments conducted and more\nanimals studied, we did generate fairly consistent food intake findings in the current,\nmodified iteration of the manuscript. Furthermore, we did observe an occasional surge or\ndip in food intake that did have to do with certain mice grinding and destroying food that\nwas then no longer measurable and excluded since the food intake findings were no\nlonger interpretable. We also now address food intake changes in the discussion.\n9) “Figure 4: adipocyte and muscle fiber CSA in healthy controls are missing. 3 mice\nper cohort for histology looks too low and unlikely to provide such statistical differences.\nI wonder whether the average CSA from each mouse has been used as a single point or\neach adipocyte/fiber area has been considered as an independent point. The second\noption is incorrect. In the third panel of Figure 4E, there is an unexpected increase in mice\ninjected with ΔGFP cells; this highlights a possible limitation of having only 3 mice per\ncohort. The same applies for Figure 5H, where there is an unexpected reduced CSA in\nΔGFP.”: We now have provided cross sectional area and muscle-stained sections for\nmultiple experiments with proper negative controls throughout the paper. From a\nhistological perspective we have also evaluated adipocyte and myocyte cross sectional\narea in Figures 3C-E, 3H, 3I, 4K-L, 4N, 6I-J, 6L-N, 7F-G, 7I-J and Supplemental\nFigures 6J-L.\n10) “Figure 6, panel B: is tumor volume at sacrifice statistically different? Tumor mass,\nanorexia, serum GDF15 and cachexia look directly correlated.”: Tumor volume at sacrifice\nis not statistically different, and neither is tumor mass among KPL groups shown in\nFigures 7A and B. We have now added an associative comparison between tumor\nvolume and cachexia factor GDF15 serum levels in Figure 1F to show that tumor volume\ndoes not associate with GDF15 serum levels. There is no statistical association between\nserum GDF15 levels and tumor size. Throughout multiple genetic silencing and antibody\nneutralization experiments we were able to disassociate tumor growth with cachexia\nphenotype (see Figures 2, 4, 5, 7, S4, S6).\n11) “In panel J form the same figure, it looks like that anti GDF15 initially promotes\nadipose mass accumulation in KPL-bearing mice, followed by a second phase of\n‘resistance/ineffectiveness’. A similar phenomenon seems to occur in mice bearing\nH1792ΔSTK11 tumors (Supplemental figure 4, panel E). Given the potential translational\nvalue of the study, the prophylactic nature of the design is not fully convincing, suggesting\nto perform a therapeutic study to show the impact of anti GDF15 in early cachectic mice\n(i.e. starting at day 15 or 20 from KPL cell injection). Similarly, a survival (or even a ‘time\nto humane endpoint’) study would strongly increase the translational value.“: GDF15\nantibody appears to block the cachexia from a preventative perspective (Figures 5A-G\nand 7) as well as new data showing antibody treatment reversal of the cachexia\nphenotype once cachexia development has initiated and progressed (see Figures 5H-\nP). During the revision of this manuscript, this same GDF15 neutralization antibody\nshowed significant beneficial effect for cancer patients with high GDF15 levels in a\npublished clinical trial (PMID: 39282907; added to our discussion) and is moving to a\nphase III randomized trial across multiple primary cancers to determine if a therapeutic\nindication can be solicited. Our data also shows that disrupting GDF15 in STK11/LKB1\nfunction-deficient NSCLC with low GDF15 levels also prevent cachexia (Figures 6 and\n7), which expands the utilization of this GDF15 antibody to patients with low circulating\nGDF15 concentrations if their tumors carry STK11/LKB1 loss-of-function mutations. We\nalso verified GDF15 antibody does not non-specifically increase body weight, fat mass,\nor lean mass by after administration to non-tumor bearing mice (Supplemental Figure\n3).\n12) “Panel K: first time showing skeletal muscle weight; I would recommend including\nhealthy controls.”: We added the appropriate healthy controls to Figures 3A, B, F, G,\nFigures 4J, M, Figures 5N, P, and all new experiments.\n13) “Similarly, relative tumor-free body weight and relative tumor-free lean mass for\nKPL + IgG/GDF15ab are missing.” Relative tumor free body weight and tumor free lean\nmass for KPL studies -/+ IgG or GDF15 antibody are now presented (Figure 7).\n14) “Figure 7: very elegant gene reconstitution experiment, however, the reduced\ntumor growth rate in reconstituted LKB1 cells does not allow to conclude on the impact\non cachexia, given that the same tumor mass developed in a different time frame likely\ninduced a differential adaptation in the hosts, resulting in a different cachectic state.”\nSTK11/LKB1 is a known tumor suppressor gene and it is not surprising that there is\ndecreased tumor growth with reconstitution. Although there is a delay in tumor growth,\nthe slope of growth is similar once the tumors actually start growing. This differential\ngrowth in Figure 8 was accounted for by only measuring wasting at equivalent tumor\nsizes. New data presented in Supplemental Figure 6 shows the dependence of the\nH1437 line on tumor-secreted GDF15 (further increasing the generalizability of our\nfindings). Reconstitution of wild-type STK11/LKB1 into this line decreased the integrated\nstress response (measured by PERK, peIF2α, ATF4, and CHOP induction) and\ndecreased production and processing of GDF15 in cell culture which is independent of\ntumor growth (Figures 8A-C).\n15) “Panel D: the x axis of the graph has to be fixed.” The axis labeling has been\ncorrected in Figure 8D.\n16) “I hardly understand the supplemental figure 5, where two experiments almost\nidentical show contrasting or even opposite results. How can 2 weeks of age difference\nin adult mice produce such different results?”: As pointed out by the reviewer, food\ngrinding was an issue with the previous data. We have now included experimentation\nwhere food grinding is not a complication of the experiment. See new Figure 8F.\n17) “The authors state that GDF15 is relevant for muscle contractility. Considering that\nin this work GDF15 signaling is blocked, an assessment of muscle functon (at least grip\ntest and/or treadmill test, in case physiological muscle strength is not a lab commodity)\nshould be included.” As requested, we have also now added grip strength studies as an\nadditional metric of muscle function in cachexia in Figures 2L and 4I and Supplemental\nFigure 2I.\n18) “Minor Comments”:\na. “Is it necessary to have 7 figures in the main text?: To address all of the\ncomments from the reviewers, we now actually have 8 figures and added\nmore supplementary figures. We did remove two figures to lessen the\nburden on the reader as pointed out by this reviewer. We removed two\nsupplementary figures that were antibody experiments in patient-derived\nlines since we already have multiple GDF15 silencing and antibody\nexperiments in multiple models. We believe the remaining figures are\nessential to the evaluation of GDF15 in multiple STK11/LKB1-mutated\nmodels to show the generalizability of our findings.\nb. “Line 221: despite having ECHO-MRI and CSA data, to state “adipose and\nmuscle atrophy”, the final tissue weights should be reported.” Based on this\nsuggestion, we have now added fat pad and muscle weights based on a\nrevised experiment for our main H1573 patient-derived cachexia model\n(Figures 3A-B, 3F-G) as well as in the new orthotopic experiments\n(Figures 4J and 4M) and male-female mouse experiment (Figures S2J-\nM).\nc. “WB: overexposed and low quality of the results.” It is unclear what figure\nthe reviewer is referencing. We went through and optimized each\nimmunoblot analysis exposure. We intentionally overexposed the GDF15\nand ATF4 blots in Figure 8B to identify changes across a range of glucose\nlevels.\nd. “Line 87, ‘STK11/LKB1 functional variant’: do the authors refer to an\noncogenic mutant?” We use the word “functional variant” to describe a\ngenetic change that is predicted to alter the function of the expressed\nprotein.\ne. “Lines 483-485: the authors propose the use of metabolic cages and single-\nhousing to better study food intake alterations associated with changes in\nGDF15 circulating levels. While single-housing can be an interesting\napproach to isolate the behavior/contribution of each animal, changing\nanimal housing numerosity can be an independent factor affecting the\nfeeding behavior, possibly determining discrepancies with the observations\npresented in this study. The authors should mention this limitation in the\nproposed future approach.: We added a section to the discussion in the\nmetabolic cage section which reads “…with results interrupted\nunderstanding singly-housed mice itself might alter food intake.”\nf. “Lines 928, 944, 959: please adjust group numerosity reported in the text\n(doesn’t match with the related graphs).”: Due to the reviewer’s comment\nthat we have too many figures, we removed the figures that were referenced\nin 944 and 959. We have appropriately corrected the animal numbers in the\nfigure legend as pointed out in line 928.\ng. Multiple clerical/wording changes: Changes made as pointed out by the\nreviewer.\nReviewer #2 Comments:\n1) “I co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in peer\nreview and to provide appropriate recognition for Early Career Researchers who co-\nreview manuscripts.”: Reviewer 2 co-reviewed this manuscript with one of the reviewers\nwho provided the listed reports. See Reviewer 1 or 3 Responses.\nReviewer #3 Comments:\n1) “The study relies predominantly on subcutaneous tumor transplantation of a large\nnumber of cells, with tumors often reaching extremely large sizes (<1cm3) before any\nmeaningful weight loss is observed. In human patients, tumor burden is often poorly\npredictive of weight loss associated with cachexia, with small tumors capable of inducing\ncachexia. A main concern over the validity of the conclusions of this manuscript is that\nthese subcutaneous tumor models are poorly reflective of human disease, and do not\naccurately represent the complex interplay between tumor genetics and the tumor\nmicroenvironment. In an autochthonous tumor model of LKB1 deficient NSCLC, where\ntumors develop in the native environment of the lung, levels of GDF15 were shown to be\nelevated as compared to LKB1 WT tumors but play no part in phenotypes associated with\ncachexia. The link between LKB1 loss and GDF15, and the role of GDF15 in promoting\ncachexia in these tumors, should be studied in models of NSCLC where tumors grow in\nthe native environment in the lung, including genetically engineered mouse models and\nsyngeneic transplant studies. Such studies will give insight as to whether the link between\nGDF15 and LKB1 loss is robust or an artifact of the model chosen.”: The subcutaneous\ntransplantation model is a well-accepted model to study cachexia. (PMID: 40298389,\nPMID: 21680814, PMID: 32661391, PMCID: PMC4224962) The subcutaneous model has\nsimilar tumorigenesis kinetics as other models, allows investigators to test multiple human-\nderived models, and enables investigators to distinguish relevant soluble molecule\nsources of cachexia-relevant factors from human (tumor) vs mouse (host). We have now\nvalidated our original findings in multiple subcutaneous xenotransplant models using\nseveral patient-derived lines. We also show that subcutaneous injection of the syngeneic\nKPL model (derived from the TP53/KRAS GEMM mouse model) into immunocompetent\nmice are also dependent on GDF15 for cachexia development (see Figures S5 and 7).\nTo further address the concerns of tumors developing in the native environment of the\nlung, we collaborated with the Dr. James Kim laboratory (see new added authors) to\nconduct an orthotopic lung model experiment. This new data is included in Figure 4.\nDespite a similar tumor burden in the lung, the GDF15-silenced tumor-bearing mice have\ndecreased serum GDF15 with a corresponding rescue of body weight, adipose tissue, lean\nmass, and muscle function, to similar levels found in mice that were not administered\ntumor. The cachexia findings from the orthotopic model correlate with similar findings from\nthe subcutaneous model, we believe strengthening the manuscript significantly.\n2) “There seems to be an intrinsic effect on neutralizing GDF15 even in tumors that\ndo not have high levels of circulating GDF15 (in particular, Supplemental Figure 4). This\nsuggests that treatment with anti-GDF15 antibody is driving the reversal of cachexia\nphenotypes rather than through blocking the actions of GDF15. To confirm an on target\neffect of GDF15 neutralization, the anti-GDF15 antibody studies should be complemented\nwith treatment of tumors with anti-GFRAL antibody, the only known receptor of GDF15.\nAdditionally, previous studies have indicated that the principal contributor of GDF15\nmediated weight loss is from sympathetic output rather than changes in food intake.\nIt would be insightful to see the effect of chemical sympathectomy by 6-OHDA treatment to\nobserve whether this output is playing a role in cachexia in these models.”: [REDACTED]\n3) “LKB1 regulates a host of downstream kinases including AMPK and the salt-\ninducible kinases (SIKs). Previous studies have indicated the tumor suppressive effects of\nLKB1 are dependent on the SIKs. Additionally, a CRTC2-CREB-SIK axis mediates the\ninflammatory potential of cells harboring loss of LKB1. It would be informative to see if\nincrease in GDF15 expression is mediated through this same pathway or if other kinases\ndownstream of LKB1 play a role.”: For additional mechanistic insights, please see\nReviewer 1 Comment 1. Additionally, we also knocked out CRTC2 or CREB in the H1437\ncachexia inducing NSCLC line and determined that the silencing of either gene failed to\nblock GDF15 production in tissue culture (data not included in manuscript). We are\ncurrently investigating other downstream signaling molecules including AMPK, SIK, and\nAMPK-related kinases that may be responsible for transducing STK11/LKB1-LOF\ncachexia signals, a focus of a subsequent manuscript.\n4) “The evidence linking lowering of GDF15 levels to STK11 reconstitution is clear in\nFigure 7, yet whether this lowering of GDF15 levels is primarily driving the improvement in\nweight loss is correlative. Studies utilizing GDF15 loss or neutralization should be\nperformed to examine the role of the molecule in this context.”: As requested by the\nreviewer, we now include data in Supplemental Figure 6 where we silence GDF15 in the\nH1437 cell line. As expected, silencing of GDF15 in this same cell line abrogates cachexia\ndevelopment without affecting tumor growth.\n5) “Minor Comments”:\na. “GDF15 is a marker of metabolic stress – thus as tumors grow larger, it is\nexpected that levels of GDF15 expression would increase as nutrient\navailability in the tumor diminishes and the metabolic demands for growth\nincrease. Thus, it would be informative for the authors to provide relative\ntumor size effects on levels of GDF15 mRNA expression and circulating\nGDF15 levels. As requested by the reviewer, we have included new Figure\n1F which directly compares tumor sizes versus serum GDF15 levels in our\npatient-derived non-cachexia-inducing and cachexia-inducing lines. There\nare no statistical associations between tumor size and serum GDF15\nlevels.\nb. “How does relative bodyweight in tumor bearing animals change over time\nwith GDF15 antibody administration? Only bodyweight at endpoint is\nprovided.”: Due to the limitation of the size of Figure 5 after adding the\nrescue experiment, we did not include the body weight change over time.\nWe have included this data for Reviewer 3’s review for both the old\nprevention experiment as well as the new rescue antibody experiment.\nThis data is not significantly different than the endpoint Relative Tumor\nFree Body Weights included in new Figure 5.\nc. “It is unclear what is being blotted for in figure 3C, and the P and M labels\nare not described. It figure 3F, it seems there is both a loss of parental and\nmouse GDF15 levels in cells targeted with a guide against GDF15.”: P\nindicates proprotein GDF15, M indicates mature GDF15; we clarified these\nabbreviations in each respective figure legend.\nd. “In the food intake studies in figure 3F, while reaching statistical\nsignificance, the differences are extremely marginal and most likely not\nrelevant to the relative weight loss.”: We agree with this assessment, and\nwe changed the corresponding result section to “…silencing of GDF15\npromoted minimal change in food intake.” Furthermore, we expanded the\nsection on food intake regulation in our discussion.\ne. “In syngeneic experiments, what is the effect of GDF15 neutralization in\nthe context of a KP tumor? This can provide more insight into the effect of\nthe GDF15 antibody in the context of low levels of GDF15.”: To address\nthe effect of GDF15 antibody on low levels of GDF15, we injected GDF15\nantibody into non-tumor bearing mice (new Figure S3). There were no\nsignificant changes in lean mass, fat mass, or body weight in mice\nreceiving the GDF15 antibody compared to control groups.\nResponse to Reviewer Referees Letter\nWe again thank the reviewers for their critiques of our revised manuscript and appreciate their\nconstructive feedback. Although Reviewer 1 felt that we addressed their major concerns,\nReviewer 3 has raised an additional concern that was not mentioned in their original critiques\nregarding the lack of food intake changes in our cancer cachexia models that are dependent on\nthe secretion of GDF15, a potential contradictory finding. We agree with Reviewer 3 that pulsatile\ninjections of recombinant GDF15 into wild-type mice over a short time frame consistently\ndemonstrate food intake decreases by other investigators (PMID: 28953886, 28846099,\n28846098, 28846097). However, we disagree that GDF15’s role in body weight loss is solely due\nto a decrease in food intake in pathological states that occur over longer intervals (PMID:\n32661391, 37380764, 26672741). For example, injections of recombinant GDF15 into\nobesity/MASH preclinical models induces body weight loss; however, the major driver of this\noutcome is through a food intake independent process (PMID: 37380764). More relevant to our\ncurrent studies, other groups have also shown that tumor cachexia models associated with and\neven dependent on elevated GDF15 do not show changes in food intake (PMID: 32661391) with\nmanipulation of circulating GDF15 concentrations. This team also directly compared a\nrecombinant GDF15 administration model to a GDF15 dependent tumor cachexia model and\nobserved that the tumor model, unlike the recombinant GDF15 administration model, did not\ndemonstrate any significant changes in food intake (see data below) with alterations in serum\nGDF15. Clinically relevant, patients with cancer cachexia with elevated serum GDF15 also did\nnot show significant differences in their appetite for food based on a self-assessment survey\n(PMID: 26672741). Therefore, our studies demonstrating a lack of change in food intake in our\ncancer cachexia models is consistent with what other groups have observed in pre-clinical models\nand clinically.\nUltimately, the scope of this manuscript was focused on linking loss-of-function mutations in\nSTK11/LKB1 in NSCLC with an increase in tumor cell integrated stress response resulting in the\ntumor secretion of the cachexia factor GDF15 promoting cachexia. The new concern of Reviewer\n3 regarding the lack of food intake changes in our tumor cachexia models relative to GDF15 could\nalso be explained by previous work we have conducted studying leptin counter-regulatory host\nmechanisms. Our group showed previously that the effects of cachexia factors on promoting\nanorexia, including those observed with IL-6 family members, are counterbalanced by\nprogrammed decreases in systemic concentrations of the pro-anorexic molecule leptin as the\nmice undergo cachexia-induced adipose wasting, culminating in efforts of the host to return food\nintake back towards unaltered baseline levels (PMID: 30046014). We provide new data in this\nrevised manuscript showing that serum leptin is also significantly reduced in our models\nconcomitant with wasting associated with elevated serum GDF15 concentrations in STK11/LKB1-\nmutated NSCLC bearing mice. The decreased serum leptin provides a possible explanation for\nwhy there is no obvious food intake changes in our STK11/LKB1-mutatant NSCLC cachexia\nmodels.\nAlthough understanding exactly how GDF15 triggers body weight loss in a food intake-\nindependent manner in the pathological condition defined by tumor bearing mice is an interesting\nquestion, we anticipate the true insight into this mechanism will be clarified in subsequent\npublications. But, as a means of satisfying the concerns raised by Reviewer 3, we now show in\nan exploratory manner that muscle sarcolipin (Sln)-mediated energy expenditure is potentially\ncritical to GDF15-supported wasting independent of changes in food intake in our preclinical\nmodels. A recent paper has highlighted this food intake independent connection between Sln and\nelevated tissue specific energy expenditure in GDF15 models (PMID: 37380764). Another recent\npublication in a mouse model of mitochondrial myopathy and body weight loss showed that\nGDF15 neutralization significantly decreases muscle Sln while rescuing body weight changes\n(PMID 39976232). A detailed description of these findings, as subfigures, and the appropriate\nreferences are detailed below with our direct response to Reviewer 3’s additional concerns. We\nhave also modified our discussion accordingly and add this new serum leptin and muscle\nsarcolipin data to our figures in an effort to satisfy Reviewer 3’s remaining concerns."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In the current revised version of the manuscript, a huge amount of work has been performed to address all my comments/ raised issues in a satisfactorily manner. The manuscript text has been extensively and consistently reorganized, now flowing and reading much better. I see potentially more limitations in the results performed following reviewer 3 comments, however I think it goes over fair peer-review / my assigned tasks.\nI only suggest minor changes in order to optimize and standardize the communication of the results.\nGrip strength assessments are now included as functional outcome. Please express the values in N (Newton) or mN representing a force, while grams represent static masses. Moreover, Animal force should be normalized by the animal body weight at the beginning of the experiment to account for the variability in animal size. The interindividual body weight variability may also impact on tissue masses, I thus suggest to use the same normalization (initial body weight) for muscle and fat masses. The normalized values may even be presented in a supplementary table, including the initial body weight along with the tissues analyzed.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "While the manuscript has been improved by experiments conducted during the revision process, major concerns remain regarding multiple mechanistic aspects of this study at present time. There is a major mechanistic gap as to why elevations in serum GDF-15 do not robustly decrease food intake in this animal model. Additionally, genetic deletion of GDF-15 in tumor cells or blockade through neutralizing antibody treatment in some cases reduces food intake, which runs in direct conflict with our current understanding how GDF-15 promotes weight loss. GDF-15 binds to its sole receptor GFRAL, which is expressed only in the area postrema and nucleus solitary tract of the brainstem, driving weight loss through reductions in food intake and peripheral sympathetic nervous system activity. In line with this understanding, treatment with the anti-GFRAL antibody should induce an increase in food intake through blockage of GDF-15’s actions in the CNS, which the authors do not observe. Furthermore, these high-circulating levels of GDF-15 should induce expression of early response genes in the brainstem, such as cFOS, which is a major omission in the manuscript. Multiple groups have identified that GFRAL expression is limited to the brain-stem and is not expressed in peripheral tissues. The lack of rescue of food intake with neutralization of GDF-15/GFRAL signaling axis suggests the rescue effect is not due to direct actions of GDF-15 but through other artifactual pathways.\nAdditionally, genetic or pharmacological silencing of GDF-15 seems to improve cachexia in every cell line and animal model utilized in this manuscript, regardless of circulating GDF-15 levels, which makes these interventions most likely artifactual. While the authors acknowledge that blockade of GDF-15 may be due to changes in other molecules, understanding how blockade of GDF-15 changes other circulating factors should be included in the manuscript to conclusively support the conclusions of the paper. The findings presented at this point remain too discordant for publication in Nature Communications.\nMinor comments:\nTreatment of healthy animals with anti-GDF-15 antibodies is not informative, as these animals possess no basal GDF-15 signaling or systemic inflammation. As requested in prior comments previous to resubmission, the authors should examine the effect of GDF-15 in an Lkb1-WT tumor where circulating levels of GDF-15 are marginal and systemic inflammation is present. Rescue in this setting would suggest an artifactual effect of antibody treatment."}, "author_response": "Reviewer #1 (Remarks to the Author):\n“In the current revised version of the manuscript, a huge amount of work has been\nperformed to address all my comments/ raised issues in a satisfactorily manner. The\nmanuscript text has been extensively and consistently reorganized, now flowing and\nreading much better. I see potentially more limitations in the results performed following\nreviewer 3 comments, however I think it goes over fair peer-review / my assigned tasks.\nI only suggest minor changes in order to optimize and standardize the communication\nof the results.”\nMinor Comments\n1) “Grip strength assessments are now included as functional outcome. Please\nexpress the values in N (Newton) or mN representing a force, while grams represent\nstatic masses. Moreover, Animal force should be normalized by the animal body\nweight at the beginning of the experiment to account for the variability in animal\nsize. The interindividual body weight variability may also impact on tissue masses,\nI thus suggest to use the same normalization (initial body weight) for muscle and\nfat masses. The normalized values may even be presented in a supplementary table,\nincluding the initial body weight along with the tissues analyzed.”\nWe have converted the values of grip strength in grams to values in N in Figures 2M, 4I, and\nS3J. We also added each mouse’s day 0 body weight, measured grip strength (g), transformed\ngrip strength (N), and normalized grip strength (N/g, grip strength/day 0 body weight) in\nSupplementary Table 2. Both transformed grip strength and normalized grip strength data show\nconsistent results that STK11/LKB1-mutated NSCLC tumor implantation decrease grip strength\ncompared to non-tumor bearing mice, while tumor GDF15 silencing rescues the phenotype.\nReviewer #3 (Remarks to the Author):\nMajor Comments\n1) “While the manuscript has been improved by experiments conducted during the\nrevision process, major concerns remain regarding multiple mechanistic aspects\nof this study at present time. There is a major mechanistic gap as to why elevations\nin serum GDF-15 do not robustly decrease food intake in this animal model.\nAdditionally, genetic deletion of GDF-15 in tumor cells or blockade through\nneutralizing antibody treatment in some cases reduces food intake, which runs in\ndirect conflict with our current understanding how GDF-15 promotes weight loss.\nGDF-15 binds to its sole receptor GFRAL, which is expressed only in the area\npostrema and nucleus solitary tract of the brainstem, driving weight loss through\nreductions in food intake and peripheral sympathetic nervous system activity. In\nline with this understanding, treatment with the anti-GFRAL antibody should\ninduce an increase in food intake through blockage of GDF-15’s actions in the CNS,\nwhich the authors do not observe. Furthermore, these high-circulating levels of\nGDF-15 should induce expression of early response genes in the brainstem, such\nas cFOS, which is a major omission in the manuscript. Multiple groups have\nidentified that GFRAL expression is limited to the brain-stem and is not expressed\nin peripheral tissues. The lack of rescue of food intake with neutralization of GDF-\n15/GFRAL signaling axis suggests the rescue effect is not due to direct actions of\nGDF-15 but through other artifactual pathways.”\nWe are in complete agreement with the reviewer's assertion that the anorexic effects of\nGDF15 represent a cornerstone of modern cachexia research. Indeed, numerous studies in both\npreclinical models and human patients have established a strong correlation between elevated\ncirculating GDF15 levels and anorexia (PMID: 28953886, 28846099, 28846098, 28846097,\n39282907). However, the regulation of food intake is a complex process, orchestrated by an\ninterplay of central and peripheral signals that govern hunger, satiation, and long-term energy\nbalance. Therefore, we respectfully argue against the over-simplified emphasis of GDF15-GFRAL\nanorexia as the only factor driving wasting in our cancer cachexia models. This perspective\noverlooks the complexity of food intake regulation and a vast body of clinical and experimental\nevidence of non-anorexia mediated GDF15 related body weight loss in pathological states. We\nagree with Reviewer 3 that pulsatile injections of recombinant GDF15 into wild-type mice over a\nshort time frame consistently demonstrates food intake decreases, as described by other\ninvestigators (see Extended Figure 4A below from Nature Medicine manuscript PMID:\n32661391). However, we disagree that GDF15’s role in body weight loss is solely due to a\ndecrease in food intake in pathological states that occur over longer/chronic windows of time\n(PMID: 32661391, 26672741, 37380764).\nThe landmark study by Suriben et al. in Nature Medicine (PMID: 32661391) demonstrated\nthat their tumor cachexia models dependent on elevated GDF15 for regulating wasting also failed\nto show changes in food intake similar to our data in our current manuscript with GDF regulation\ngenetically and/or pharmacologically. The investigators implanted HT1080 tumor cells into mice\nand showed that the mice undergo GDF15-dependent cachexia (see extended Figure 3a in their\nmanuscript). There was not a significant difference in food intake in these animals when compared\nto non-tumor bearing mice (see extended Figure 4B; included above). The authors also directly\ncompared the mouse model of administered recombinant GDF15 (Extended Figure 4A; included\nabove) versus their GDF15-dependent cancer cachexia model (Extended Figure 4B; included\nabove) to show that the cancer cachexia model wasting was independent of net food intake\ndifferences. Although the pulsatile injection of GDF15 caused a significant decrease in food intake,\nthe tumor bearing mice that ultimately succumbed to GDF15-dependent cachexia did not have a\nchange in food intake. The authors further developed a monoclonal antibody that acted as a\ntherapeutic antagonistic,, 3P10, targeting GDF15’s receptor GFRAL. When the authors\nadministrated 3P10 to HT1080 tumor bearing mice, the drug rescued body weight loss (Extended\nFigure 4C; included above). Similar to our models, there was also a significant rescue in adipose\nand lean mass tissue loss (Extended Figures 4D and 4F; included above). In this rescue model,\nhowever, the investigators did not observe a significant change in food intake in the 3P10\ntreatment cohort (Extended Figure 4E;\nincluded above). These findings of a lack of\nfood intake changes in the treatment of their\nGDF15-dependent cancer cachexia models\nare concordant with our genetically-silenced\nand pharmacologically inhibited cachexia\nmodels outlined in this manuscript\nsuggesting that there are other mechanisms\nin play in these pathologic tumor states that\nresist or counterbalance the anorexic affects\n(see below for a plausible mechanism)\nAdditionally, and more clinically relevant,\nGarcia and colleagues measured serum\ninflammatory markers including GDF15 in\nnon-cancer patient controls (59) and cancer\npatients with (n=58) and without (n=72)\nweight loss (Journal of Cachexia,\nSarcopenia, and Muscle, PMID: 26672741).\nOf the cancer patients, lung cancer was the\nmost common primary tumor (see their\nTable 1; included on the left). Serum GDF15\nwas significantly elevated in cancer patients\nwith weight loss compared to those cancer\npatients without weight loss and non-cancer\ncontrols (See Figure 1 in the manuscript).\nThe cancer patients with weight loss also had\nsignificant decrements in fat mass, lean\nmass, and muscle function as judged by grip\nstrength (see their Table 1; included on the\nleft). These patient characteristics are similar\nto those that we observe in our GDF15-\ndependent STK11/LKB1-mutated patient-\nderived tumor bearing mice. Importantly, the\ncancer patients with weight loss also did not have a significant difference in food intake as judged\nby appetite scores (see their Table1; included on previous page).\nMore recently, there is growing evidence in\nother pathological conditions that the majority\nof GDF15’s weight loss effect is independent of\nfood intake. Injections of recombinant GDF15\ninto obesity/MASH models induce body weight\nloss; however, the majority of this weight loss is\ndue to a food intake independent process\n(Nature, PMID: 37380764). To prove this, the\ninvestigators pair-fed animals and matched\nfood intake to cohorts of mice that received two\ndoses of GDF15 (see Figure 1D; included on\nthe right). The reduced food intake only\naccounted for half of the body weight changes\nobserved in their MASH model. The\ninvestigators were able to show that increased\nmuscle energy expenditure through elevated\nmuscle sarcolipin (Sln) accounted for the\nmajority of the food intake independent body\nweight loss in their GDF15 injection\nadministration model. This finding provides\nanother example of how GDF15 can promote\nweight loss without ultimately influencing net\nfood intake.\nIn another set of studies, the PolgD257A/D257A\n(PLOG) mouse – a mitochondrial myopathy\nmouse model with decreased body weight, fat\nmass and fat-free mass phenocopying\ncachexia characteristics, showed elevated\ncirculating GDF15 levels. Antibody\nneutralization of this GDF15 rescued mice from\nthis phenotype, concordant with decreases in\nmuscle Sln expression (J Cachexia Sarcopenia\nMuscle, PMID: 39976232). These two findings\nsuggest that GDF15 could induce food-intake\nindependent body weight loss by increasing\nmuscle Sln resulting in increased muscle\nenergy expenditure. Neutralization of GDF15\nreverses body weight loss and at the same time\nsignificantly decreases muscle Sln.\nFIGURE 5 | GDF15 neutralization alleviated the\nabnormal gene expression profile in POLG\ngastrocnemius muscle. (B) Volcano plot of\ndifferentially expressed genes in POLG- GDF15\nmAB2 versus POLG-Veh gastrocnemius muscle,\nfollowing transcriptomic analysis. n = 8/group.\nTo evaluate if muscle Sln expression, a surrogate for muscle energy expenditure, associated with\nbody weight loss in our STK11/LKB1-mutated cachexia models dependent on tumor secreted\nGDF15, we analyzed Sln mRNA levels by qPCR in both gastronomic and soleus muscles from\nthese animal cohorts. In our isogenic H1573 subcutaneous male cancer cachexia model, Sln\nexpression level negatively correlated with body weight, as tumor-bearing mice with parental\nH1573 or H1573ΔGFP (intact GDF15) had a higher Sln expression in gastronomic muscle (see\nFigure 3J in our revised manuscript) and soleus muscle (see Figure S2 in our revised manuscript)\ncompared to non-tumor (PBS) bearing mice and mice bearing H1573 ΔGDF15, which also correlated\nwith increasing muscle loss/atrophy.\nTaken together, our data is consistent with what other investigators have observed in\npathological states, especially in their cancer cachexia pre-clinical models. Furthermore, the lack\nof food intake changes is also observed in patients with cancer-associated weight loss with\nelevated GDF15. In our opinion, our findings of a lack of change in food intake in our GDF15-\ndependent STK11/LKB1-mutated cancer cachexia models are consistent with what others have\nobserved in the field.\n2) “Additionally, genetic or pharmacological silencing of GDF-15 seems to improve\ncachexia in every cell line and animal model utilized in this manuscript, regardless\nof circulating GDF-15 levels, which makes these interventions most likely\nartifactual.”\nTo generalize our findings, we conducted our experiments using several patient-derived and\nsyngeneic NSCLC lines in immunocompromised and immunocompetent mice, respectively. We\nalso completed our studies in both male and female mice. Additionally, we conducted our studies\nboth in subcutaneous and orthotopic models. We not only genetically silenced GDF15 from\nmultiple patient-derived lines to show that the host cachexia was suppressed, we also targeted\nGDF15 with neutralizing antibody in these models to further demonstrate the contribution of\nGDF15 to the cachexia phenotype. Reviewer 3’s original critique stated “to confirm an on-target\neffect of GDF15 neutralization, the anti-GDF15 antibody studies should be complemented with\ntreatment of tumors with anti-GFRAL antibody, the only known receptor of GDF15.” Therefore, to\nconfirm an on-target effect of GDF15 neutralization, we obtained GFRAL antibody and confirmed\nthat this administration also blocks the GDF15-dependent STK11/LKB1-mutated NSCLC host\nwasting as suggested by Reviewer 3. In our opinion, this comprehensive approach decreases the\npossibility that these findings are artificial and instead it increases rigor and generalizability of our\nstudies.\n3) “While the authors acknowledge that blockade of GDF-15 may be due to changes\nin other molecules, understanding how blockade of GDF-15 changes other\ncirculating factors should be included in the manuscript to conclusively support\nthe conclusions of the paper. The findings presented at this point remain too\ndiscordant for publication in Nature Communications.”\nWe previously reported that tumor-secreted cachexia factors (such as leukemia inhibitory\nfactor LIF) could cause significant loss of adipose tissue, muscle atrophy, and anorexia\n(PMID: 30046014) over time in preclinical models. At early longitudinal time points of these\npublished tumor cachexia experiments for this PMID, the wild-type C57/BL6 mice injected with\ncachexia factor LIF demonstrated an early transient state of hypophagia that returned to\nnormophagia as leptin levels decreased as a compensation to the initial adipose loss from the\ncytokine cachexia signals (PMID: 30046014). This reduction in serum leptin concentrations, which\ntranslated into a temporary signal promoting net food intake, counterbalanced the cachexia factor\nanorexia effect. Administration of cachexia factors into ob/ob (leptin deficient) and db/db (leptin\nreceptor deficient) mice led to a perpetual change in food intake due to lack of this host\ncompensatory mechanism. These findings and parallel ones observed in the current submitted\nmanuscript ultimately describe why one does not always observe net reduced food intake in tumor\ncachexia models, likely due to host compensation for the elevated serum GDF15 with a reduction\nin serum leptin.\nTo specifically test if this host counterregulatory mechanism is occurring in our STK11/LKB1-\nmutated cachexia models that are dependent on secreted GDF15, we analyzed the serum leptin\nlevels in multiple experiments presented in the manuscript. In our isogenic H1573 subcutaneous\nmale cachexia mouse model, tumor bearing mice with parental H1573 or H1573ΔGFP had a\nsignificant decrease in their serum leptin concentrations compared to non-tumor (PBS) bearing\nmice (see Figure 2F in the revised manuscript). H1573ΔGDF15 tumor bearing mice, which did not\nexhibit the cachexia phenotype, had serum concentrations of leptin similar to that of non-tumor\n(PBS) bearing mice. In our isogenic H1573 subcutaneous female mouse model (see Figure S3E\nin the revised manuscript), we observed a similar trend of decreased serum leptin concentrations\nin mice injected with H1573 cell lines with GDF15 intact. Serum leptin concentrations were\nbaseline in female H1573ΔGDF15 bearing mice. We also observed altered serum leptin levels in our\northotopic experiment (see Figure S8A in the revised manuscript) and antibody neutralization\nexperiment (see Figure S8B in the revised manuscript) with H1573 cell lines commiserate with\nthe level of adipose wasting, and finally in parallel experiments conducted leveraging the H1437\ncell line (see Figure S8C in the revised manuscript). In all these experiments, animals\nadministered the STK11/LKB1-mutated NSCLCΔGDF15 lines or parental line treated with GDF15\nantibody demonstrated a suppressed cachexia phenotype concomitant with a reversal of the\nreductions in serum leptin concentrations, approaching the control concentrations observed in\nmice bearing STK11/LKB1-mutated NSCLC ΔGFP lines or treated with IgG. This data is\nconsistent with our previous published findings that decreased leptin can counterbalance the\ndecreased food intake changes induced by cachexia factors. This data likely explains the\ninconsistency of the anorexia phenotype observed in these GDF15-dependent cancer cachexia\nmodels.\nMinor comments:\n1) “Treatment of healthy animals with anti-GDF-15 antibodies is not informative, as\nthese animals possess no basal GDF-15 signaling or systemic inflammation. As\nrequested in prior comments previous to resubmission, the authors should\nexamine the effect of GDF-15 in an Lkb1-WT tumor where circulating levels of GDF-\n15 are marginal and systemic inflammation is present. Rescue in this setting would\nsuggest an artifactual effect of antibody treatment.”\nThe experiment proposed by the reviewer is subjecting wild-type STK11/LKB1 tumor bearing mice\nwith GDF15 neutralization antibody to see if there is a “rescue” of the phenotype. We note that\nneither healthy animals nor STK11/LKB1-WT tumor-bearing mice develop cachexia, rendering\nthe hypothetical \"rescue\" in the suggested experiment irrelevant to our findings. Wild-type\nSTK11/LKB1 tumor bearing mice do not exhibit a cachexia phenotype to “rescue” since they are\nsimilar to non-tumor bearing mice in regard to body weight and composition. However, we\nappreciate the reviewer’s concern for an “artifactual effect”. Therefore, we have carefully designed\nour experiments to mitigate this risk of an “artifactual effect” of our findings. Our comprehensive\napproach included: 1) Genetic manipulation; we utilized CRISPR/Cas9 to knock out GDF15 in\nseveral patient-derived NSCLC cell lines (H1573, H1437, H2122, H1944). The resulting isogenic\ncell lines, lacking GDF15 protein expression, demonstrated a rescue of cancer cachexia in both\nmale and female mice as well as in subcutaneous and orthotopic models. 2) Pharmacological\ninhibition; we employed a GDF15 antibody to neutralize GDF15 in both syngeneic mouse\nmodels and patient-derived NSCLC (H1573) cancer cachexia models through both preventative\nand rescue experimental paradigms. We also injected these antibodies into non-tumor bearing\nmice and did not see any changes in body weight and composition. 3) Genetic rescue and\npathway validation; in an STK11/LKB1-mutated NSCLC cell line (H1437), we restored wild-type\nSTK11/LKB1. This intervention not only decreased GDF15 production but also prevented cancer\ncachexia, strongly supporting a key role for the loss-of-function STK11/LKB1 variant - GDF15\ninduction pathway as important to NSCLC patients with cachexia. 4) Specificity confirmation;\nas recommended, we performed a GFRAL neutralization antibody experiment using our tumor\ncachexia mouse models. These findings further confirmed that the observed effects we are seeing\nare specifically mediated by GDF15 signaling within our STK11/LKB1-mutated NSCLC cancer\ncachexia model. Collectively, these diverse experimental strategies provide strong evidence that\nour observations are biologically significant and not attributable to antibody artifacts or offsite\neffects."}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors have provided some additional experiments and published work to suggest that perhaps blockade of GDF15 or its receptor GFRAL can prevent wasting without impacting feeding. Although this is an intriguing hypothesis, there is still a major gap on how this is happening and although the authors site prior studies, they need to address this mechanistic gap. Given that GFRAL is only expressed in the brainstem, the assumption is that the observed effects of wasting in relation to GDF15 are occuring through the brainstem without food intake being affected. If that is the case the authors should assess neuronal activity by c-FOS staining in the brainstem area postrema (AP) in response to GDF15 or GFRAL blockade. If they do not see any effects in AP activity that suggests that there is GDF15/GFRAL signaling in another tissue/area which they should define. Is there GFRAL expression in the muscle or the fat?"}, "author_response": "Response to Referees\nWe again thank the reviewers for their critiques of our second revised manuscript and once\nagain appreciate their constructive feedback. We believe this manuscript has greatly improved\ndue to the critiques provided by all reviewers and the additional experimentation completed to\naddress these critiques. Reviewers 1 and 2 had no additional critiques. Reviewer 3 recognizes\nthat our data as well as other investigators’ data demonstrate that blockade of GDF15 can prevent\nwasting independent of food intake changes in cancer cachexia models. However, they also\nwould like this mechanistic gap to be addressed. We did provide additional data to support\ncompensatory mechanisms by the mouse (i.e. changes in serum leptin and muscle sarcolipin\nlevels) to maintain the food intake levels in our previous revision. We also believe that the exact\nmechanism will require a substantial body of work that is outside of the scope of this current\nmanuscript and therefore will be addressed in a future manuscript. We believe we have completed\nthe scope of this current manuscript which focused on linking loss-of-function mutations in\nSTK11/LKB1 in NSCLC with an increase in tumor cell integrated stress response resulting in the\ntumor secretion of the factor GDF15 promoting cachexia.\nReviewer #3 (Remarks to the Author):\nMajor Comments\n“The authors have provided some additional experiments and published work to\nsuggest that perhaps blockade of GDF15 or its receptor GFRAL can prevent wasting\nwithout impacting feeding. Although this is an intriguing hypothesis, there is still a\nmajor gap on how this is happening and although the authors site prior studies, they\nneed to address this mechanistic gap. Given that GFRAL is only expressed in the\nbrainstem, the assumption is that the observed effects of wasting in relation to GDF15\nare occuring through the brainstem without food intake being affected. If that is the\ncase the authors should assess neuronal activity by c-FOS staining in the brainstem\narea postrema (AP) in response to GDF15 or GFRAL blockade. If they do not see any\neffects in AP activity that suggests that there is GDF15/GFRAL signaling in another\ntissue/area which they should define. Is there GFRAL expression in the muscle or the\nfat?”\nThe scope of this current manuscript focuses on linking loss-of-function mutations in STK11/LKB1\nin NSCLC with an increase in tumor cell integrated stress response resulting in the tumor\nsecretion of the factor GDF15 promoting cachexia. We agree there is still a mechanistic gap with\ntargeting GDF15 or its receptor GFRAL to prevent wasting that seems to be independent of food\nintake changes. We provided both compensatory mechanisms (decrease in serum leptin) as well\nas GDF15 food intake independent mechanisms (increase of muscle sarcolipin levels) to provide\nalternative explanations for the lack of observed food intake changes with GDF15 blockade in\ncancer cachexia models observed by our group and other groups. Although a detailed\nunderstanding of this GDF15 mechanism is warranted to uncover its biological relevance\nindependent of cancer cachexia, we believe this will require enough experimentation to support\nits own manuscript which we plan to proceed with in the future. We had already detailed in the\ndiscussion our future “plan to use syngeneic model systems to characterize STK11/LKB1-mutant\ntumor-derived GDF central and peripheral effects leveraging tissue specific knockout models of\nthe GDF15 receptor, GFRAL” which will ultimately address Reviewer 3’s comment and suggestion."}]

Decision letter