bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Genetic Factors Associated with Absolute and Relative Plasma Concentrations of Calcitriol
Authors: Wilson Robin Taylor , Safford Susan E. , Ostrom Quinn T. , Wang Ming , McDonald Alicia C. , Salzberg Anna C. , Barnholtz-Sloan Jill S. , Richie John P.
Published: May 2023

AbstractBackground:
Little is known regarding factors associated with calcitriol and a relative measure of calcitriol, the calcitriol-24,25-dihydroxyvitamin D3-calcifediol proportion ratio (C24CPR).
Methods:
Using a cross-sectional study design, healthy young adults of African and European descent, matched (1:1) on age (±5 years) provided a blood sample in non-summer months (N = 376). Vitamin D metabolites were measured in plasma with HPLC/MS-MS. West African genetic ancestry proportion (WGA) was estimated using STRUCTURE modeling of genetic ancestry-informative markers. Multivariable regression models were used to estimate the association of WGA and vitamin D–pathway gene variants with calcitriol and C24CPR, controlling for days from summer solstice, age, sex, blood pressure, body mass index, dietary vitamin D intake, oral contraceptive/medroxyprogesterone acetate use, smoking, tanning bed use, and time of day.
Results:
Calcitriol and C24CPR were not highly correlated (rho = 0.14), although both were significantly, positively, and monotonically associated with WGA (Ptrend 0.025 and <0.001, respectively). In fully adjusted models, genetic factors explained a greater proportion of variability in C24CPR (R2 = 0.121 and 0.310, respectively). Variants in genes with associated with calcitriol (CALB1, CYP27B1, GC, and PPARGC1A) differed from those associated with C24CPR (CYP3A43, FGF23, KL, and VDR).
Conclusions:
Both absolute and relative measures of calcitriol were significantly higher among African Americans. Otherwise, these biomarkers appear to be genetically distinct.
Impact:
C24CPR may be better suited to personalized medicine, due to a higher proportion of population variability explained by genetic variation and a less skewed distribution.
Data from Estimating the Number of Men Living with Metastatic Prostate Cancer in the United States
Authors: Devasia Theresa P. , Mariotto Angela B. , Nyame Yaw A. , Etzioni Ruth
Published: May 2023

AbstractBackground:
Metastatic prostate cancer (MPC) includes metastases detected at diagnosis (de novo) and those occurring after diagnosis with early-stage disease (recurrent). Cancer registries collect data only on de novo MPC, providing a partial picture of the burden of MPC. We use cancer registry data to estimate the number of men living with MPC in the United States including both de novo and recurrent cases.
Methods:
We apply a back-calculation method to estimate MPC incidence and prevalence from U.S. prostate cancer mortality and de novo MPC relative survival for cases diagnosed between 2000 and 2017 in 18 Surveillance, Epidemiology, and End Results registries. We hold overall prostate cancer mortality and MPC survival constant for future prevalence projections.
Results:
On January 1, 2018, we estimated 120,400 U.S. men living with MPC (45% de novo, 55% recurrent). The age-adjusted prevalence in 2018 for Black men was over double that of White men (137.1 vs. 62.2 per 100,000 men). By 2030, 192,500 men are expected to be living with MPC, with the increase being driven by population growth projections.
Conclusions:
The number of men living with MPC in the United States exceeds 100,000 and represents a small fraction of the >3 million men living with a prior diagnosis of prostate cancer.
Impact:
Relatively similar fractions of de novo and recurrent MPC among prevalent cases highlight opportunities for management of localized disease in reducing the MPC burden. Changes in diagnostic technologies could lead to greater growth in MPC cases in the United States than projected.

See related commentary by Stopsack et al., p. 585
Supplementary Figure S3 from Integrating Tumor-Intrinsic and Immunologic Factors to Identify Immunogenic Breast Cancers from a Low-Risk Cohort: Results from the Randomized SweBCG91RT Trial
Authors: Stenmark Tullberg Axel , Sjöström Martin , Niméus Emma , Killander Fredrika , Chang S. Laura , Feng Felix Y. , Speers Corey W. , Pierce Lori J. , Kovács Anikó , Lundstedt Dan , Holmberg Erik , Karlsson Per
Published: May 2023

The tumor-intrinsic model, Proliferative Index, could predict the prognostic effect from the immunological model, Immunescore. With high Proliferative Index values, Immunescore was strongly prognostic. However, with low Proliferative Index values, trends toward the opposite were observed.
Supplemental Table S1 from BRD9 Degradation Disrupts Ribosome Biogenesis in Multiple Myeloma
Authors: Kurata Keiji , Samur Mehmet K. , Liow Priscilla , Wen Kenneth , Yamamoto Leona , Liu Jiye , Morelli Eugenio , Gulla Annamaria , Tai Yu-Tzu , Qi Jun , Hideshima Teru , Anderson Kenneth C.
Published: May 2023

Univariate and multivariate analysis of BRD9 expression
Supplementary Figures S1-S17 from Zinc Finger MYND-Type Containing 8 (ZMYND8) Is Epigenetically Regulated in Mutant Isocitrate Dehydrogenase 1 (IDH1) Glioma to Promote Radioresistance
Authors: Carney Stephen V. , Banerjee Kaushik , Mujeeb Anzar , Zhu Brandon , Haase Santiago , Varela Maria L. , Kadiyala Padma , Tronrud Claire E. , Zhu Ziwen , Mukherji Devarshi , Gorla Preethi , Sun Yilun , Tagett Rebecca , Núñez Felipe J. , Luo Maowu , Luo Weibo , Ljungman Mats , Liu Yayuan , Xia Ziyun , Schwendeman Anna , Qin Tingting , Sartor Maureen A. , Costello Joseph F. , Cahill Daniel P. , Lowenstein Pedro R. , Castro Maria G.
Published: May 2023

Supplementary Figures and Figure Legends
Supplementary Figure S4 from NPY Methylated ctDNA is a Promising Biomarker for Treatment Response Monitoring in Metastatic Colorectal Cancer
Authors: Janssens Katleen , Vanhoutte Greetje , Lybaert Willem , Demey Wim , Decaestecker Jochen , Hendrickx Koen , Rezaei Kalantari Hassan , Zwaenepoel Karen , Pauwels Patrick , Fransen Erik , Op de Beeck Ken , Van Camp Guy , Rolfo Christian , Peeters Marc
Published: May 2023

Median methylation ratio.
Metastatic Prostate Cancers with BRCA2 versus ATM Mutations Exhibit Divergent Molecular Features and Clinical Outcomes
Authors: Hwang Justin , Shi Xiaolei , Elliott Andrew , Arnoff Taylor E. , McGrath Julie , Xiu Joanne , Walker Phillip , Bergom Hannah E. , Day Abderrahman , Ahmed Shihab , Tape Sydney , Makovec Allison , Ali Atef , Shaker Rami M. , Toye Eamon , Passow Rachel , Lozada John R. , Wang Jinhua , Lou Emil , Mouw Kent W. , Carneiro Benedito A. , Heath Elisabeth I. , McKay Rana R. , Korn W. Michael , Nabhan Chadi , Ryan Charles J. , Antonarakis Emmanuel S.
Published: May 2023

Purpose:

In patients with metastatic prostate cancer (mPC), ATM and BRCA2 mutations dictate differences in PARPi inhibitor response and other therapies. We interrogated the molecular features of ATM- and BRCA2-mutated mPC to explain the divergent clinical outcomes and inform future treatment decisions.

Experimental Design:

We examined a novel set of 1,187 mPCs after excluding microsatellite-instable (MSI) tumors. We stratified these based on ATM (n = 88) or BRCA2 (n = 98) mutations. As control groups, mPCs with mutations in 12 other homologous recombination repair (HRR) genes were considered non-BRCA2/ATM HRR-deficient (HRDother, n = 193), whereas lack of any HRR mutations were considered HRR-proficient (HRP; n = 808). Gene expression analyses were performed using Limma. Real-world overall survival was determined from insurance claims data.

Results:

In noncastrate mPCs, only BRCA2-mutated mPCs exhibited worse clinical outcomes to AR-targeted therapies. In castrate mPCs, both ATM and BRCA2 mutations exhibited worse clinical outcomes to AR-targeted therapies. ATM-mutated mPCs had reduced TP53 mutations and harbored coamplification of 11q13 genes, including CCND1 and genes in the FGF family. BRCA2-mutated tumors showed elevated genomic loss-of-heterozygosity scores and were often tumor mutational burden high. BRCA2-mutated mPCs had upregulation of cell-cycle genes and were enriched in cell-cycle signaling programs. This was distinct from ATM-mutated tumors.

Conclusions:

Tumoral ATM and BRCA2 mutations are associated with differential clinical outcomes when patients are stratified by treatments, including hormonal or taxane therapies. ATM- and BRCA2-mutated tumors exhibited differences in co-occurring molecular features. These unique molecular features may inform therapeutic decisions and development of novel therapies.
Supplementary Table S5 from BAP1 Loss Is Associated with Higher ASS1 Expression in Epithelioid Mesothelioma: Implications for Therapeutic Stratification
Authors: Barnett Sarah E. , Kenyani Jenna , Tripari Martina , Butt Zohra , Grosman Rudi , Querques Francesca , Shaw Liam , Silva Luisa C. , Goate Zoe , Marciniak Stefan J. , Rassl Doris M. , Jackson Richard , Lian Lu-Yun , Szlosarek Peter W. , Sacco Joseph J. , Coulson Judy M.
Published: May 2023

Patient scoring and survival summary
Supplementary Methods and References from BAP1 Loss Is Associated with Higher ASS1 Expression in Epithelioid Mesothelioma: Implications for Therapeutic Stratification
Authors: Barnett Sarah E. , Kenyani Jenna , Tripari Martina , Butt Zohra , Grosman Rudi , Querques Francesca , Shaw Liam , Silva Luisa C. , Goate Zoe , Marciniak Stefan J. , Rassl Doris M. , Jackson Richard , Lian Lu-Yun , Szlosarek Peter W. , Sacco Joseph J. , Coulson Judy M.
Published: May 2023

Supplementary Methods, References and List of Figures and Tables
Supplementary Table 1 from Bone Marrow Mesenchymal Stem Cells Induce Metabolic Plasticity in Estrogen Receptor–Positive Breast Cancer
Authors: Buschhaus Johanna M. , Rajendran Shrila , Chen Siyi , Wharram Bryan L. , Bevoor Avinash S. , Cutter Alyssa C. , Humphries Brock A. , Robison Tanner H. , Farfel Alex P. , Luker Gary D.
Published: May 2023

1. Quantified metabolic shifts in response to antiestrogenic therapy and MCT1/4 inhibition. Change in median values in picoseconds of NADH or CFP lifetime (ATP) after addition of each compound. Labels at the top of the table denote the relevant comparison in each column.
Figure S3 from Bone Marrow Mesenchymal Stem Cells Induce Metabolic Plasticity in Estrogen Receptor–Positive Breast Cancer
Authors: Buschhaus Johanna M. , Rajendran Shrila , Chen Siyi , Wharram Bryan L. , Bevoor Avinash S. , Cutter Alyssa C. , Humphries Brock A. , Robison Tanner H. , Farfel Alex P. , Luker Gary D.
Published: May 2023

Figure S3. ROS quantification for MSCs in monoculture and co-culture. Flow cytometry data for ROS detected by CellROX Green in HS5 and HS27a MSCs in monoculture or co-culture with MCF7 (A), T47D (B), or HCC1428 (C) ER+ breast cancer cells. Solid and dashed lines depict MSCs in monoculture co-culture, respectively. Cell type of interest is bolded in co-culture comparisons. N>10,000 cells.
Figure S3 from The Elongin BC Complex Negatively Regulates AXL and Marks a Differentiated Phenotype in Melanoma
Authors: Schieven Sebastiaan M. , Traets Joleen J.H. , Vliet Alex v. , Baalen Martijn v. , Song Ji-Ying , Guimaraes Marcos Da Silva , Kuilman Thomas , Peeper Daniel S.
Published: May 2023

Extended data on Figure 3: Elongin B/C regulate AXL independently of hypoxia.
Data from Comparative Tumor Microenvironment Analysis of Primary and Recurrent Ovarian Granulosa Cell Tumors
Authors: Khlebus Eleonora , Vuttaradhi Veena K. , Welte Thomas , Khurana Namrata , Celestino Joseph , Beird Hannah C. , Gumbs Curtis , Little Latasha , Legarreta Alejandra Flores , Fellman Bryan M. , Nguyen Tri , Lawson Barrett , Ferri-Borgogno Sammy , Mok Samuel C. , Broaddus Russell R. , Gershenson David M. , Futreal P. Andrew , Hillman R. Tyler
Published: May 2023

Abstract
Adult-type granulosa cell tumors (aGCT) are rare ovarian sex cord tumors with few effective treatments for recurrent disease. The objective of this study was to characterize the tumor microenvironment (TME) of primary and recurrent aGCTs and to identify correlates of disease recurrence. Total RNA sequencing (RNA-seq) was performed on 24 pathologically confirmed, cryopreserved aGCT samples, including 8 primary and 16 recurrent tumors. After read alignment and quality-control filtering, DESeq2 was used to identify differentially expressed genes (DEG) between primary and recurrent tumors. Functional enrichment pathway analysis and gene set enrichment analysis was performed using “clusterProfiler” and “GSVA” R packages. TME composition was investigated through the analysis and integration of multiple published RNA-seq deconvolution algorithms. TME analysis results were externally validated using data from independent previously published RNA-seq datasets. A total of 31 DEGs were identified between primary and recurrent aGCTs. These included genes with known function in hormone signaling such as LHCGR and INSL3 (more abundant in primary tumors) and CYP19A1 (more abundant in recurrent tumors). Gene set enrichment analysis revealed that primarily immune-related and hormone-regulated gene sets expression was increased in recurrent tumors. Integrative TME analysis demonstrated statistically significant depletion of cancer-associated fibroblasts in recurrent tumors. This finding was confirmed in multiple independent datasets.
Implications:
Recurrent aGCTs exhibit alterations in hormone pathway gene expression as well as decreased infiltration of cancer-associated fibroblasts, suggesting dual roles for hormonal signaling and TME remodeling underpinning disease relapse.
Abstract A004: KRAS allelic imbalance drives an epithelial MAPK-dependent tumor initiation program that is inefficient in provoking metastasis in colorectal cancer in vivo
Authors: Najumudeen Arafath K. , Fey Sigrid K. , Campbell Andrew D. , Sansom Owen J.
Published: May 2023

Allelic imbalance is reported to be a frequent event in cancers and a common feature associated with oncogenes such as KRAS and BRAF. However the functional and therapeutic consequences of such imbalances are poorly understood. Mutations in the KRAS oncogene are one of the most prevalent events in human cancers and heterozygous KRAS mutations are well-described functionally and are thus viewed as sufficient for tumorigenesis. Recent evidence shows high incidence of KRAS gene dosage changes in human cancers, including loss of the normal wild-type allele of KRAS. However, there is still much debate over the function of wild-type KRAS in tumour initiation, progression and therapy response. Using advanced genetically engineered mouse models of colorectal cancer (VillinCRE Apcfl/fl; LSL-KrasG12D/+ (AK), LSL-KrasG12D/+; Trp53fl/fl; Rosa26N1iCD/+ (KPN)) we investigated the functional impact of wild-type Kras in oncogenic Kras driven tumour initiation (AK), progression and metastasis (KPN) in vivo. Mechanistically, wild-type Kras restrains oncogenic Kras signalling and significantly affects the efficiency of the oncogenic Kras induced transformation and response to therapy. We demonstrate a suppressive role for wild-type Kras during tumorigenesis and highlight the critical impact of wild-type Kras upon therapeutic response and tumour progression in Kras mutant CRC. Wild-type KRAS-deficient colorectal tumours are characterized by increased MAPK signalling and transcriptional activation of MAPK regulators. Importantly, loss of wild-type Kras in oncogenic KRAS-driven aggressive KPN tumours significantly alter tumour progression and liver metastasis, showing increased immune infiltration and WNT signalling. In addition, loss of wild-type Kras modulates response to therapeutic intervention and sensitizes wild-type Kras deficient tumours to MEK1/2 inhibition. This study demonstrates a suppressive role for wild-type Kras during tumour initiation and highlights the critical impact of wild-type Kras upon therapeutic response to MAPK and tumour progression in KRAS mutant CRC.

Citation Format: Arafath K. Najumudeen, Sigrid K. Fey, Andrew D. Campbell, Owen J. Sansom. KRAS allelic imbalance drives an epithelial MAPK-dependent tumor initiation program that is inefficient in provoking metastasis in colorectal cancer in vivo [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A004.
Abstract A026: Stochastic phenotypes in RAS-dependent developmental diseases
Authors: Marmion Robert A. , Simpkins Alison G. , Barrett Lena A. , Schottenfeld-Roames Jodi , Schüpbach Trudi , Shvartsman Stanislav Y.
Published: May 2023

Germline mutations upregulating RAS signaling are associated with multiple developmental disorders. A hallmark of these conditions is that the same mutation may present vastly different phenotypes in different individuals, even in monozygotic twins. We demonstrate how the origins of such largely unexplained phenotypic variations may be dissected using highly controlled studies in Drosophila that have been gene edited to carry activating variants of MEK, a core enzyme in the RAS pathway. This allowed us to measure the small but consistent increase in signaling output of such alleles in vivo. The fraction of mutation carriers reaching adulthood was strongly reduced, but most surviving animals had normal RAS-dependent structures. We rationalize these results using a stochastic signaling model and support it by quantifying cell fate specification errors in bilaterally symmetric larval trachea, a RAS-dependent structure that allows us to isolate the effects of mutations from potential contributions of genetic modifiers and environmental differences. We propose that the small increase in signaling output shifts the distribution of phenotypes into a regime, where stochastic variation causes defects in some individuals, but not in others. Our findings shed light on phenotypic heterogeneity of developmental disorders caused by deregulated RAS signaling and offer a framework for investigating causal effects of other pathogenic alleles and mild mutations.

Citation Format: Robert A. Marmion, Alison G. Simpkins, Lena A. Barrett, Jodi Schottenfeld-Roames, Trudi Schüpbach, Stanislav Y. Shvartsman. Stochastic phenotypes in RAS-dependent developmental diseases [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A026.
Abstract A029: Unfolding the role of cell state on stress granule heterogeneity and function in KRAS-driven pancreatic cancer
Authors: Redding Alexandra , Fonteneau Guillaume , Grabocka Elda
Published: May 2023

Phenotypic and functional heterogeneity is a common feature of cancer cells that poses as a major therapeutic challenge. Previous work has implicated the phase-separation organelles, stress granules (SGs), as important mediators of tumorigenesis that function as a mutant KRAS-driven stress-adaptive mechanism to enhance cellular fitness. We have found that mutant KRAS pancreatic cancer cells show a high degree of intercellular heterogeneity in SG formation, ranging from no SGs to markedly high levels. Given the function of SGs in cancer cell fitness and the role of cancer cell heterogeneity in tumor progression and therapeutic response, we sought to understand the determinants of SG heterogeneity and its role in KRAS-driven tumorigenesis. Here, we show that the capability of KRAS mutant pancreatic cancer cells to form SGs is dependent on the cell cycle state, with cells in G2 phase showing markedly enhanced SG formation. We demonstrate that this heterogeneity in SG formation is determined by a cell cycle specific interplay between the activator of cell death, Caspase 3, and the SG regulator molecule 15-deoxy-delta-12,14-prostaglandin J2 (15-d-PGJ2). We show that 15-d-PGJ2 production in G2 is driven by calcium-dependent phospholipase A2 (cPLA2) activity and is subject to regulation by Caspase 3. Importantly, we show that heterogeneity in SG levels translates into a functional heterogeneity whereby SG inhibition preferentially sensitizes cells in G2 to stress stimuli. Altogether these data support a model whereby SG formation integrates cues from cell death and proliferative signals and indicate that SG levels may be critical to achieve a clear signal for cell cycle progression at the G2/M border. Furthermore, our findings suggest that KRAS-driven cancer cells may show differential sensitivity to anti-SG therapy as they progress through the cell cycle, and pairing such therapy with G2-synchronizing chemotherapeutics may increase susceptibility and thus treatment efficacy.

Citation Format: Alexandra Redding, Guillaume Fonteneau, Elda Grabocka. Unfolding the role of cell state on stress granule heterogeneity and function in KRAS-driven pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A029.
Abstract A036: Oncogenic mutations in BRAF and MEK weaken the ATP-stabilized inactive conformation of RAF to promote RAF dimerization and MAPK pathway activation
Authors: Wendorff Timothy J. , Kung Jennifer , Sudhamsu Jawahar
Published: May 2023

The RAS-RAF-MEK-ERK signaling axis is upregulated in many human cancers due to its central role in cell growth, differentiation and survival. Oncogenic transformation frequently arises from RAS, BRAF and MEK mutations that activate the pathway. In the clinic, similar transformations also recur as resistance mutations to KRAS.G12C inhibitor treatment. BRAF mutations at V600 (Class I) activate BRAF by releasing the kinase’s activation segment and rendering BRAF activity dimerization-independent. Yet, it remains unclear how a diverse set of dimerization-dependent BRAF mutations (Class II/III) activate the pathway. MEK mutations are assumed to bypass the RAF node entirely. We previously reported a 2.9-Å-resolution crystal structure of human BRAF kinase domain (BRAFKD) in complex with MEK and the ATP analog AMP-PCP, revealing interactions between BRAF and ATP that stabilize an inactive, pre-signaling monomeric conformation of BRAFKD and explain how ATP breaks RAF dimers in solution. Surprisingly, the β3-αC loop of MEK provides additional stabilizing interactions to the BRAF-bound AMP-PCP molecule. Utilizing an in vitro RAF dimerization assay, we find that MEK not only modulates RAF-RAF dimerization, it also enhances both the dimer-breaking effect ATP and the dimer-forming effect of paradoxical activator molecules, such as GDC-0879. Structural analysis reveals that all common oncogenic BRAF mutations alter key interactions with ATP that stabilize the inactive monomer conformation. We find ATP is unable to break RAF dimers containing Class I, II or III mutations, confirming these mutants counteract the inhibitory effects of ATP binding by lowering the threshold for RAF dimerization and thus pathway activation. We further find that oncogenic MEK mutants, in particular β3-αC loop deletions, favor RAF-RAF dimerization, even in the presence of ATP. Our study establishes a framework for rationalizing oncogenic BRAF mutations, and suggests that oncogenic MEK mutants can act, at least in part, by promoting RAF dimerization. This model provides a better understanding of activation mechanisms in the context of constantly evolving resistance mutations where oncogenesis continues to depend on the MAPK pathway.

Citation Format: Timothy J. Wendorff, Jennifer Kung, Jawahar Sudhamsu. Oncogenic mutations in BRAF and MEK weaken the ATP-stabilized inactive conformation of RAF to promote RAF dimerization and MAPK pathway activation [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A036.
Abstract B003: Combined inhibition of the MAPK and Hippo pathways drives efficacious tumor suppression in a faithful model of mutant Kras (KPC) PDAC
Authors: Chakravarti Deepavali , Muller Florian , Gyuris Jeno , Baloglu Erkun , Kunnimalayiaan Selvi , Olson Jill , Shacham Sharon
Published: May 2023

Oncogenic mutations in the KRAS oncogene hyperactivate the MAPK pathway, a key driver of oncogenesis in the majority of human malignancies. KRAS-driven tumors are aggressive and highly refractory to standard-of-care treatments, thus intense efforts have been devoted to developing inhibitors of its activity. However, pioneering studies in engineered conditional mKras driven mouse models of pancreatic ductal adenocarcinoma (KPC) have demonstrated that while mKras is necessary for tumor maintenance, the anti-neoplastic effects of mKras extinction can be counteracted by activation of the Hippo pathway. These data strongly suggest that the full realization of MAPK pathway (including KRAS) inhibitors’ clinical potential requires the concomitant suppression of the function of YAP/TEAD, the effectors of Hippo pathway. Here, we directly test this hypothesis utilizing Sporos BioDiscovery’s next generation TEAD inhibitor (SPR1) - optimized to maximize anti-neoplastic activity and minimize toxicity through fine-tuning of TEAD paralog specificity. Using mKras murine (KrasG12D mutant, p53 null, PDAC) and human cell lines - we demonstrated strong interaction between SPR1 TEAD inhibitors and the clinical MEK inhibitors (since no G12D inhibitor is yet approved) Trametinib/Binimetinib in vitro. Functional TEAD inhibition was also shown by the reduced expression of the YAP/TEAD transcriptional targets CTGF and CYR61. Further, using a well-established murine orthotopic model, KPC (KrasG12D;p53-/-;p48Cre), that faithfully mimics human PDAC, we demonstrated robust combined efficacy of Trametinib and SPR1 TEAD inhibitors in vivo in suppressing tumor growth and strikingly doubling the survival time of the tumor-bearing mice. To our knowledge, this is the first preclinical demonstration of the therapeutic potential of a MAPK + TEAD inhibitor combination in a mKras G12D-driven orthotopic PDAC; given the high barrier for efficacy of this KPC model –we believe these results favorably presage generalization to MAPK/TEAD inhibitor combinations in other cancer setting and translatability to the clinic.

Citation Format: Deepavali Chakravarti, Florian Muller, Jeno Gyuris, Erkun Baloglu, Selvi Kunnimalayiaan, Jill Olson, Sharon Shacham. Combined inhibition of the MAPK and Hippo pathways drives efficacious tumor suppression in a faithful model of mutant Kras (KPC) PDAC [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B003.
Abstract B009: TEAD inhibition overcomes YAP1/TAZ-driven resistance to RAS inhibitors in KRASG12C-mutant cancers
Authors: Edwards Alexander C. , Stalnecker Clint A. , Morales Alexis J. , Taylor Khalilah E. , Hallin Jill , Olson Peter , Tang Tracy T. , Engstrom Lars , Post Leonard , Christensen James G. , Cox Adrienne D. , Der Channing J.
Published: May 2023

Activated mutants of KRAS comprise the major oncogenic drivers in lung (LAC), colorectal (CRC), and pancreatic ductal (PDAC) adenocarcinoma. Recent success in covalently targeting one KRAS mutant (KRASG12C) led to FDA approval of the first anti-KRAS therapy (G12Ci). However, both primary and treatment-induced acquired resistance to G12Ci have been observed. While analyses of relapsed patients have identified reactivation of the key KRAS effector signaling network as a driver of resistance, the mechanisms in ~50% of patients are not known. To identify potential resistance mechanisms, we applied a CRISPR-Cas9 loss-of-function screen targeting the druggable genome. In addition to genes discovered recently in relapsed patients (e.g., PTEN, NF1), we also identified loss of multiple components of the Hippo tumor suppressor pathway as drivers of G12Ci resistance (NF2, LATS1/2, TAOK1/2 and STK3/4). We therefore determined if activation of the functionally related transcriptional co-activators, YAP1 and TAZ, normally inhibited by Hippo signaling, can drive resistance to G12Ci. We first determined that ectopic expression of constitutively activated YAP1/TAZ was sufficient to impair the anti-proliferative and pro-apoptotic effects of G12Ci treatment in KRASG12C-mutant LAC, CRC, and PDAC cell lines. Conversely, genetic suppression of YAP1/TAZ enhanced G12Ci sensitivity. YAP1/TAZ requires association with TEAD and other transcription factors to regulate transcription. We determined that YAP1/TAZ mutants deficient in TEAD binding failed to drive resistance to G12Ci treatment. Further supporting a role for TEAD, both overexpression of a TEAD dominant negative mutant and treatment with pan-TEAD pharmacological inhibitors phenocopied the effects of YAP1/TAZ genetic suppression and sensitized KRASG12C mutant cancer cells to G12Ci. Finally, transcriptional analyses support a model where YAP1/TAZ-TEAD overcomes KRASG12C addiction by restoring a subset of KRAS-dependent gene transcription. In summary, our observations support YAP1/TAZ-TEAD signaling as a novel driver of resistance to KRAS inhibition and support the use of TEAD inhibitors to enhance the anti-tumor efficacy of KRAS-targeted therapies.

Citation Format: Alexander C. Edwards, Clint A. Stalnecker, Alexis J. Morales, Khalilah E. Taylor, Jill Hallin, Peter Olson, Tracy T. Tang, Lars Engstrom, Leonard Post, James G. Christensen, Adrienne D. Cox, Channing J. Der. TEAD inhibition overcomes YAP1/TAZ-driven resistance to RAS inhibitors in KRASG12C-mutant cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B009.
Abstract B013: Exploring the switch II pocket of KRAS(G12D) with mutant-selective monobody inhibitors
Authors: Hattori Takamitsu , Akkapeddi Padma , Khan Imran , Koide Akiko , Glasser Eliezra , Ketavarapu Gayatri , Whaby Michael , Zuberi Mariyam , Teng Kai Wen , Lefler Julia , Maso Lorenzo , Bang Injin , Ostrowski Michael C. , O’Bryan John P. , Koide Shohei
Published: May 2023

The G12D mutation is among the most common KRAS mutations associated with cancer, in particular pancreatic cancer. Here, we have developed monobodies, small synthetic binding proteins, that are highly selective to KRAS(G12D) over KRAS(wild type) and other oncogenic KRAS mutations, as well as over HRAS(G12D). Crystallographic studies revealed that, similar to other KRAS mutant-selective inhibitors, the initial monobody bound to the S-II pocket, the groove between switch II and alpha 3 helix. Strikingly, the monobody captured a shape of the S-II pocket distinct from previously reported structures. Unlike other G12D-selective polypeptides reported to date, the monobody used its backbone NH group to directly recognize the side chain of RAS Asp12. This feature closely resembles that of a small-molecule KRAS(G12D) inhibitor, MRTX1133. The monobody also directly interacted with H95, a residue not conserved in HRAS. These features rationalize the high selectivity toward the G12D mutant and the KRAS isoform. Structure-guided affinity maturation resulted in monobodies with low nM KD values. Deep mutational scanning of a G12D-specific monobody generated hundreds of functional and nonfunctional single-point mutants, from which we identified crucial residues for binding and those that contributed to the selectivity toward the GTP- and GDP-bound states. When expressed in cells as genetically encoded reagents, these monobodies engaged selectively with KRAS(G12D) and inhibited KRAS(G12D)-mediated signaling and tumorigenesis. These results demonstrate the feasibility of targeting KRAS(G12D) functions using a mutant selective inhibitor and will guide the development of next-generation KRAS(G12D)-selective inhibitors.

Citation Format: Takamitsu Hattori, Padma Akkapeddi, Imran Khan, Akiko Koide, Eliezra Glasser, Gayatri Ketavarapu, Michael Whaby, Mariyam Zuberi, Kai Wen Teng, Julia Lefler, Lorenzo Maso, Injin Bang, Michael C. Ostrowski, John P. O’Bryan, Shohei Koide. Exploring the switch II pocket of KRAS(G12D) with mutant-selective monobody inhibitors [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B013.
Abstract B018: The development of macrocyclic peptide Ras inhibitors
Authors: Josephson Kristopher , Silvestri Anthony , Fribourgh Jennifer L. , Bray Walter M. , David Brian , Hanna Cameron C. , Chakka Sai Kumar , Hyatt Alex , Porcella Monique , Dieppa-Matos Dio , Pye Cameron R. , Schwochert Joshua
Published: May 2023

While there has been tremendous progress developing small molecule covalent K-Ras G12C inhibitors that bind Ras-off (GDP) and inhibit nucleotide exchange, there remains a need for additional K-Ras inhibitors with different Ras isoform/mutant selectivity profiles and Ras-on (GTP) mechanisms of inhibition. Of particular interest are compounds with a pan-K-Ras profile that inhibit multiple oncogenic mutants and wild type K-Ras, but achieve a therapeutic window by sparing H and N-Ras. In addition, addressing cancers driven by K-Ras oncogenic mutants beyond G12C, K-Ras amplification, or upstream dysregulation where K-Ras is maintained in the GTP-bound state will require “Ras-on” compounds that prevent effector binding. We were interested in the potential of peptide macrocycles that bind the Switch II pocket of K-Ras, in a different conformation than the small molecules, to inhibit Ras-on by allosterically blocking Raf and other effector binding. KRpep-2d is a 19-residue disulfide bridged peptide macrocycle discovered by phage display against K-Ras G12D bound to GDP. Due to its high molecular weight (2561 Da), polarity and metabolic instability, KRpep2d is inactive in in vitro cell assays. And while biochemical experiments have shown that it binds the Ras-off GDP-bound conformation of K-Ras with high affinity to inhibit nucleotide exchange, it has low affinity for K-Ras GTP and does not allosterically inhibit Raf binding. Using parallel macrocyclic peptide chemistry, we made 180 analogs of KRpep-2d to explore truncation of the exocyclic residues and replacement of the labile disulfide bond. UNP-6752, the best compound from this exploratory library, had K-Ras affinities similar to KRpep2d with a 50-fold K-Ras GDP/GTP preference, but was only 11 residues and cyclized with a non-reducible linker. Critically this compound lacked the eight arginine residues of KrPep-2d, which both preclude passive-cell permeability and induce mast cell degranulation. A second iteration of 350 compounds designed to explore single or multiple amino acid substitutions in the core of UNP-6752 led to compound UNP-7572 with sub-micromolar affinity for K-Ras GTP and an IC50 in a K-Ras-Raf TR-FRET assay of ~800 nM. Working from the UNP-7572 we are tuning selectivity for K-Ras and our progress further improving allosteric Ras-Raf inhibition, cell permeability and activity will be presented.

Citation Format: Kristopher Josephson, Anthony Silvestri, Jennifer L. Fribourgh, Walter M. Bray, Brian David, Cameron C. Hanna, Sai Kumar Chakka, Alex Hyatt, Monique Porcella, Dio Dieppa-Matos, Cameron R. Pye, Joshua Schwochert. The development of macrocyclic peptide Ras inhibitors [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B018.
Abstract B023: Inhibition of RAS signaling and tumorigenesis through targeting novel vulnerabilities
Authors: Kahn Imran , Koide Akiko , Zuberi Mariyam , Ketavarapu Gayatri , Denbaum Eric , Teng Kai Wen , Rhett J. Matthew , Spencer-Smith Russell , Hobbs G. Aaron , Camp Earnest Ramsay , Koide Shohei , O'Bryan John P.
Published: May 2023

RAS GTPases are important mediators of oncogenesis with nearly 30% of human tumors harboring mutant RAS proteins. However, pharmacological inhibition of RAS has proven challenging. We have employed Monobody technology to discover novel vulnerabilities in RAS that can be exploited to inhibit RAS signaling and tumorigenesis. Monobodies are single-domain synthetic binding proteins that achieve levels of affinity and selectivity similar to antibodies. In contrast to antibodies, Monobodies are fully functional in the reducing environment of the cytoplasm and thus are particularly suitable as genetically encoded “tool biologics”. We previously developed the NS1 Monobody that inhibited RAS by targeting the α4-α5 allosteric lobe to prevent RAS self-association and nanoclustering, and NS1 has become a widely used tool in the RAS research community. Following on this success, we sought to identify additional vulnerabilities in RAS. Based on our discovery that nucleotide-free RAS (apoRAS) inhibits PIK3C2B function, we assessed the feasibility of selectively targeting this state of RAS as an approach to inhibit oncogenic RAS function. Here, we have developed several Monobodies and extensively characterized one of them, R15. Although NS1 was agnostic to the nucleotide state of RAS, R15 bound exclusively to the apo state of all three RAS isoforms but did not interact with nucleotide-loaded RAS. When expressed in cells, R15 selectively inhibited the signaling and transforming activity of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., “fast exchange mutants”), such as G13D and Q61L. Surprisingly, R15 bound and inhibited RAS(G12D) mutants which are not reported to be fast exchange. Biochemical studies demonstrated that RAS captured with R15 from cell lysates was indeed nucleotide free, suggesting that R15 traps apoRAS and prevents nucleotide reloading. Finally, intracellularly expressed R15 selectively inhibited the tumor forming capacity of human cell lines driven by fast exchange RAS mutants but not RAS(G12V) in mouse xenografts. Thus, in contrast to conventional wisdom, our approach has established a new opportunity to selectively inhibit certain RAS mutants by targeting the apo state of RAS with drug-like molecules.

Citation Format: Imran Kahn, Akiko Koide, Mariyam Zuberi, Gayatri Ketavarapu, Eric Denbaum, Kai Wen Teng, J. Matthew Rhett, Russell Spencer-Smith, G. Aaron Hobbs, Earnest Ramsay Camp, Shohei Koide, John P. O'Bryan. Inhibition of RAS signaling and tumorigenesis through targeting novel vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B023.
Abstract B024: Combined inhibition of farnesyltransferase and MEK is effective in fusion-negative HRAS-mutant rhabdomyosarcoma
Authors: Odeniyide Patience , Lisok Alla , Fertig Elana J. , Skaist Alyza , Pratilas Christine A.
Published: May 2023

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, and RAS pathway mutations are the known driver mutations in the majority of fusion-negative (FN) RMS. Studies have demonstrated that HRAS mutations are enriched in infants with FN-RMS and can be associated with inferior outcomes. Using HRAS-mutant RMS models, we have demonstrated that tipifarnib (farnesyl transferase inhibitor, FTI) decreases ERK signaling and decreases in vitro and in vivo tumor growth. The effects of tipifarnib can be incomplete, and limitations may be due to adaptive or acquired resistance, or the presence of concurrent mutations that limit the response to single agents. These features suggest that HRAS-mutated FN-RMS may be sensitive to inhibition with combination therapy that prevents or delays the emergence of resistance. We examined the transcriptional effects of tipifarnib in FN-RMS cell lines using bulk RNA-sequencing to identify therapeutic vulnerabilities that may be exploited by RAS-directed therapies. Analysis of RNA sequencing data revealed downregulation of ERK transcriptional output genes upon treatment with tipifarnib, confirming the role of the MEK-ERK pathway in mediating the response to FTI. Trametinib (MEKi) inhibits tumor growth in xenograft models of FN-RMS but has only modest activity as a single agent. The efficacy of inhibition with FTI and MEKi has not been previously explored in RAS-driven FN-RMS. We therefore tested tipifarnib in combination with trametinib in HRAS-mutant FN-RMS cell lines and utilized in vitro assays to evaluate the effects of the combination on cell growth, differentiation and signaling via RAS effector pathways. In HRAS-mutant cells, we observed additive dose-dependent 2D and 3D growth inhibition in response to the combination. Further, tipifarnib and trametinib more potently reduced ERK phosphorylation than either drug individually, indicating effective RAS pathway inhibition. Additionally, we found that the combination of tipifarnib and trametinib induced myosin heavy chain expression, suggesting both inhibition of proliferation and promotion of myogenic differentiation. In an in vivo model, the combination of tipifarnib and trametinib led to tumor regression which was not seen with either drug alone. Our data suggest that the combination of tipifarnib and trametinib is active in models of HRAS-driven FN-RMS and may represent an effective therapeutic strategy for a genomically-defined subset of patients with FN-RMS.

Citation Format: Patience Odeniyide, Alla Lisok, Elana J. Fertig, Alyza Skaist, Christine A. Pratilas. Combined inhibition of farnesyltransferase and MEK is effective in fusion-negative HRAS-mutant rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B024.
Abstract B031: Inhibition of mutant RAS via myotubularin related protein 7-mimicking peptide
Authors: Saar Daniel , Weidner Philip , Burgermeister Elke , Kragelund Birthe B.
Published: May 2023

The coiled coil domain of Myotubularin Related Protein 7 (MTMR7) and a peptide of the same sequence (M7CC) have been shown to have an anti-cancer effect via direct interaction with PPARγ (Weidner et al. 2020). The same studies also found an inhibition of ERK-mediated inactivation of PPARγ by the same peptide. In this work we have followed up on our previous findings and show a direct effect on mutant RAS activity, mediated by MTMR7 and M7CC. In the presence of MTMR7 or M7CC, a drop of active RAS-levels along with a delocalization from the plasma membrane can be observed. This goes along with a significant drop in the levels of phosphorylated ERK and the expression of RAS-controlled genes. This led to a decrease of cell proliferation in cell culture in different cancer cell lines and of tumor volume in mouse models. The effect of MTMR7 or M7CC on RAS levels was shown to be mediated by a direct interaction with RAS. Using biophysical studies including solution-state NMR spectroscopy, the interaction was in vitro shown to happen with the globular domain of K-Ras4B. Our results support a model for MTMR7 as part of a novel RAS-regulation machinery, and highlights MTMR7 as promising scaffold to be exploited for peptide-based cancer treatment. References: Weidner, Philip; Söhn, Michaela; Schroeder, Torsten; Helm, Laura; Hauber, Veronika; Gutting, Tobias et al. (2020): Myotubularin-related protein 7 activates peroxisome proliferator-activated receptor-gamma. In: Oncogenesis 9 (6), S. 59. DOI: 10.1038/s41389-020-0238-8.

Citation Format: Daniel Saar, Philip Weidner, Elke Burgermeister, Birthe B. Kragelund. Inhibition of mutant RAS via myotubularin related protein 7-mimicking peptide [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B031.
Abstract IA01: Targeting KRAS: Light at the end of the tunnel
Authors: Barbacid Mariano
Published: May 2023

KRAS, an oncogene responsible for at least one fourth of all human cancers, was discovered four decades ago. Yet, the first KRAS selective inhibitor could not be approved until 2021. In spite of this long-awaited breakthrough, approved KRAS inhibitors only block one of the multiple oncogenic isoforms, KRASG12C, an isoform primarily present in lung adenocarcinomas. Moreover, tumor resistance to these KRAS inhibitors is becoming a significant issue in the clinic (Awad et al., N Engl J Med, 2021; Zhao et al., Nature 2021). To provide experimental information that might help to improve current KRAS targeting strategies, we have generated two experimental mouse tumor models of Kras-driven lung adenocarcinoma to compare the therapeutic consequences of eliminating, instead of inhibiting, KRAS oncoproteins. In addition, we have interrogated the molecular mechanisms responsible for the induction of resistance in sotorasib treated KrasG12C/p53 mouse lung adenocarcinomas. We have also investigated the potential role of tampering with KRAS signaling pathways to develop alternative therapeutic strategies against KRAS mutant lung and pancreatic cancers. Previous studies have shown that ablation of RAF1, either by itself, or in combination with the expression of a CDK4 kinase dead isoform, induce significant levels of tumor regression in Kras/p53 driven-lung adenocarcinomas without inducing significant toxicities (Sanclemente et al. Cancer Cell, 2018; Esteban-Burgos et al., PNAS 2020). However, Kras/p53 driven-pancreatic ductal adenocarcinomas (PDAC) are completely resistant to RAF1 ablation, unless RAF1 is deleted in combination with EGFR, an upstream KRAS effector (Blasco et al, Cancer Cell 2019). Yet, ablation of RAF1 and EGFR only resulted in the complete regression of a fraction of PDAC tumors. We have now identified activation of STAT3 as the main mechanism responsible for the resistance of PDAC tumors to RAF1/EGFR ablation. Indeed, combined ablation of RAF1/EGFR/STAT3 completely blocked proliferation of all tested mouse tumor cell lines and organoids. Moreover, all tumors tested in orthotopic allograft models regressed completely with no evidence of tumor regression for at least two months of TMX exposure. Finally, I will discuss potential strategies to pharmacologically validate this triple therapeutic strategy as well as plans to combine it with current KRAS inhibitors.

Citation Format: Mariano Barbacid. Targeting KRAS: Light at the end of the tunnel [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA01.
Abstract IA15: Coordinated transcriptional and catabolic programs support iron dependent adaptation to RAS-MAPK pathway inhibition in pancreatic cancer
Authors: Perera Rushika M.
Published: May 2023

The mechanisms underlying metabolic adaptation of pancreatic ductal adenocarcinoma (PDA) cells to pharmacological inhibition of RAS-MAPK signaling are largely unknown. Using transcriptome and chromatin immunoprecipitation profiling of PDA cells treated with the MEK inhibitor, Trametinib (MEKi), we identify transcriptional antagonism between c-MYC and the master transcription factors for lysosome gene expression, the MiT/TFE proteins. Under baseline conditions, c-MYC and MiT/TFE factors compete for binding to lysosome gene promoters to fine-tune gene expression. Treatment of PDA cells or patient organoids with MEKi leads to c-MYC downregulation and increased MiT/TFE-dependent lysosome biogenesis. Quantitative proteomics of immunopurified lysosomes uncovered reliance on ferritinophagy, the selective degradation of the iron storage complex ferritin, in MEKi treated cells. Ferritinophagy promotes mitochondrial iron-sulfur cluster protein synthesis and enhanced mitochondrial respiration. Accordingly, suppressing iron utilization sensitizes PDA cells to MEKi, highlighting a critical and targetable reliance on lysosome-dependent iron supply during adaptation to KRAS-MAPK inhibition.

Citation Format: Rushika M. Perera. Coordinated transcriptional and catabolic programs support iron dependent adaptation to RAS-MAPK pathway inhibition in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA15.