bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5611: CRB-601: A highly potent and selective integrin αvβ8 blocking antibody with anti-tumoral properties
Authors: Kolodziej Andrew , Haines Eric , Morse Richard , Zhukovsky Eugene
Published: June 2022

TGFβ is a secreted protein produced by tumors that promotes cancer progression primarily via the suppression of both the innate and adaptive immune systems. This makes TGFβ a promising immunotherapeutic target in cancer. It is ubiquitously expressed in a latent (L-TGFβ) form and the latent form has been shown to promote an immune suppressive phenotype within the tumor microenvironment. Integrin αvβ8 specifically binds to L-TGFβ. This interaction is essential for the activation of L-TGFβ-mediated signals in a variety of immune cell types. Interestingly, it has been recently shown that integrin αvβ8-mediated TGFβ activation can active directly through L-TGFβ and does not require the release of active TGFβ (1). Inhibition of integrin αvβ8-mediated TGFβ activation has been shown to block immunosuppressive regulatory T cell differentiation and enhance the recruitment of cytotoxic T cells into the tumor microenvironment (2). Here, we demonstrate by Surface Plasmon Resonance (SPR) that CRB-601, our selective inhibitor of integrin αvβ8 monoclonal antibody candidate, has a high affinity and specificity for the integrin αvβ8 complex. Moreover, in comparison to competitor molecules, such as ADWA-11, CRB-601 substantially blocks TGFβ activation in a reporter cell assay system. Additionally, using syngeneic mouse models, we evaluated the anti-tumoral properties of CRB-601 as a monotherapy, as well as in combination with immune checkpoints therapies. Findings from this study highlight the importance of integrin αvβ8 blockade in mediating the immune landscape within the tumor and leads to an enhanced response to immune checkpoint therapy. In conclusion, CRB-601 is a potent and selective integrin αvβ8 blocking monoclonal antibody that enhances the activity of immune checkpoint inhibitors in vivo and holds promise as a potential combination partner for immunotherapy. Investigational New Drug (IND) enabling studies are currently underway.

References: 1: Campbell MG. et al. (2020) Cyro-EM reveals integrin-mediated TGF-β activation without release from latent TGF-β. Cell 180, 490-501. 2: Mariathasan S. et al. (2018) TGFbeta attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells. Nature 554, 544-48.

Citation Format: Andrew Kolodziej, Eric Haines, Richard Morse, Eugene Zhukovsky. CRB-601: A highly potent and selective integrin αvβ8 blocking antibody with anti-tumoral properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5611.
Abstract 5618: Multi-armed myxoma virus has therapeutic potential for treatment of multiple myeloma
Authors: de Matos Ana L. , Dutcher Rachel , Franco Lina S. , Torres-Dominguez Lino E. , Hrach Heather , Abrantes Mario , Abdullahi Wazir , Tacner Zachary , McCrite Dillon , Grigiatis Nicole , St. Peter Jack , Sharp Leslie L.
Published: June 2022

Oncolytic viruses (OV) selectively replicate in and lyse tumor cells and provide stimulation to the immune system, representing a promising therapeutic option in development to treat cancers that do not respond well to treatment with immune checkpoint inhibitors. Myxoma virus (MYXV) is a member of the Pox family of double stranded DNA viruses and infects cancer cell lines of humans and other species. The genome of MYXV is relatively large and is amenable to engineering for expression of transgenic proteins, making it an excellent oncolytic virus for introduction of immunomodulatory proteins. We generated MYXV carrying IL-12 and decorin. IL-12 is an immune modulator that activates T- and NK-cells. Cellular responses to decorin include tumor cell intrinsic signaling effects, tumor matrix modulation, and inhibition of the TGF-beta pathway. We demonstrate the activity of multi-armed MYXV against human and mouse multiple myeloma (MM) cancer cell lines. Efficacy and mechanism of action of multi-armed myxoma virus was also demonstrated in vivo via syngeneic and xenograft models. Multi-armed MYXV represents a promising therapeutic option for MM patients that do not respond well to immunotherapy.

Citation Format: Ana L. de Matos, Rachel Dutcher, Lina S. Franco, Lino E. Torres-Dominguez, Heather Hrach, Mario Abrantes, Wazir Abdullahi, Zachary Tacner, Dillon McCrite, Nicole Grigiatis, Jack St. Peter, Leslie L. Sharp. Multi-armed myxoma virus has therapeutic potential for treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5618.
Abstract 5621: NCG-X mouse: A novel animal model to evaluate preclinical studies of humanized erythroid reconstitution without irradiation
Authors: Gu Tingting , Ju Cunxiang , Sun Hongyan , Gao Xiang , Zhang MingKun , Yu Weiwei , Wang Mengting , Li Shuai , Li Shuai , Wang Huiyi , Zhao Jing
Published: June 2022

Hematopoietic stem cell (HSC) transplantation is an adoptive immunotherapy for treating malignant hematopoietic cancers and autoimmune disorders. Preclinical studies of such novel therapies require animal models that enable and sustain human erythrocyte reconstitution. Existing mouse models typically require irradiation to remove mouse HSCs from the bone marrow prior to human immune system reconstitution. Irradiation adversely affects the health of the animal by promoting necrosis and apoptosis of gastrointestinal, neural and muscle tissues, which can lead to wasting, infection, and even death. An irradiation-free mouse model for human erythroid reconstitution is in urgent need. KIT proto-oncogene receptor tyrosine kinase, also known as c-KIT, is an important cell surface marker expressed in both mouse and human HSCs, multipotent progenitors (MPP), and common myeloid progenitors (CMP). KIT signaling plays a key role in cell survival, proliferation, and differentiation. Alterned forms of this protein have been shown to be associated with some types of cancer. Point mutation of Val831Met is known to cause macrocytic anemia. We speculate that mice with this mutation experience spontaneous anemia and inhibited HSC function, which would support human erythroid reconstitution without irradiation. NCG-X mouse was generated based on this hypothesis, building upon the severe immunodeficient NCG generated by GemPharmatech years ago. This new strain of mice are deficient of T/B/NK cells with functional inhibition of HSCs. Here, we report that NCG-X supports human HSC transplantation without radiation, enabling complete avoidance of adverse effects on animal health. When compared with irradiated NCG mice, NCG-X mice are able to not only rebuild the human immune system, but also enable human erythroid cells to reconstruct the bone marrow for a prolonged period of time after engraftment: 16 and 30 weeks post-engraftment have been assayed. Though reduced red blood cells and hemoglobin have been observed, 30 weeks post-engraftment enables consistent support from the start to the finish of the preclinical phase. In conclusion, NCG-X provides an improved and preclinical relevant mouse model for therapeutic development of leukemia, thalassemia, other types of blood diseases and autoimmune diseases.

Citation Format: Tingting Gu, Cunxiang Ju, Hongyan Sun, Xiang Gao, MingKun Zhang, Weiwei Yu, Mengting Wang, Shuai Li, Shuai Li, Huiyi Wang, Jing Zhao. NCG-X mouse: A novel animal model to evaluate preclinical studies of humanized erythroid reconstitution without irradiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5621.
Abstract 5673: The role of NMDA receptors subunits in the progression of inflammatory breast cancer (IBC)
Authors: Santacruz Laura L. Mendez , Bittman-Soto Xavier S. , Rodriguez-Martir Keishla M. , Escalona Jesus M. Padilla , Peterson Esther , Maldonado-Vlaar Carmen
Published: June 2022

Inflammatory Breast Cancer (IBC) has a 73% incidence of metastasis to the brain as compared to other cancer types like breast, lung, and bone cancer. Previous research showed that tumor cells from different breast cancer subtypes (-HR/-HER2) and (-HR/+ HER2) have a high incidence of crossing the blood-brain barrier and inducing increased expression of glutamate receptors NMDA subunits (NMDAR1 and NMDR2) in the brain. IBC is the most aggressive and rare type of breast cancer. The absence of a solid tumor is replaced by swelling, redness, and skin changes, resulting in many cases in misdiagnosis of an infection in the invasion and migration phase; this type of cancer blocks blood and lymphatic vessels in the breast's skin, causing local inflammation and rapid metastasis. However, little is known of the role that these glutamatergic receptors have in the development and/or progression of IBC. The present work provides a new understanding of the cellular and molecular effects NMDA receptors (NMDAR) have on oncogenic phenotypes in IBC. The detection and quantification of the NMDAR were evaluated by RT-qPCR, Western blot, and immunofluorescence (IF) techniques in deferments breast cancer cell lines (MCF7, MDA-MB-231) and IBC cell lines (SUM149, and SUM190). Also, NMDAR antagonists (memantine and dizocilpine "MK-801") were used in 2D and 3D cell cultures to see the pro-oncogenic effects of migration and proliferation in cell models. In addition to this, the autocrine regulation of Glutamate in IBC cell lines is verified. Results showed a significant increase in the expression of NMDARs in IBC cell lines. In the functional cell culture assays with NMDAR antagonists, a decrease in migration and proliferation in IBC cell lines was found. These findings may be related to the high aggressiveness and the high rate of metastasis by IBC within the brain. Our results have high clinical relevance since having extensive knowledge of the role of NMDARs in IBC progression could complement therapeutic approaches for patients with IBC.

Citation Format: Laura L. Mendez Santacruz, Xavier S. Bittman-Soto, Keishla M. Rodriguez-Martir, Jesus M. Padilla Escalona, Esther Peterson, Carmen Maldonado-Vlaar. The role of NMDA receptors subunits in the progression of inflammatory breast cancer (IBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5673.
Abstract 5675: Pathological crosstalk between osteocytes and breast cancer cells in bone metastasis
Authors: Adhikari Manish , Sabol Hayley , anloague Aric , Khan Sharmin , Delgado-Calle Jesus
Published: June 2022

Bone provides an ideal niche for breast cancer cells (BCa) to reside and proliferate and is a frequent site of BCa metastasis. Osteocytes are the most abundant skeletal cells and permanent residents of bone. However, the role of osteocytes in BCa bone metastasis remains understudied. Here, we investigated the crosstalk between metastatic BCa cells and osteocytes. In vitro cultures revealed that conditioned media (CM) derived from metastatic E0771 BCa cells decreased by 50% the number of alive MLO-A5 and MLO-Y4 osteocyte-like cell lines. BCa cells modestly increased osteocyte apoptosis (2% vs. 6%, alone vs. exposed to BCa, respectively). Treatment with DEVD, a caspase 3 inhibitor, blocked osteocyte apoptosis but did not prevent the decrease in osteocytes induced by BCa. Thus, we studied osteocyte senescence, a process that causes irreversible cell cycle arrest and profound changes in gene expression. We found that BCa-CM upregulated the expression of markers of senescence markers (p16, p21) and senescence-associated secretory phenotype (Mmp13, Mmp9, Vcam-1) in osteocyte-like cell lines. Similarly, BCa-CM increased the expression of senescent markers in ex vivo bone organ cultures containing primary osteocytes. Osteocytes increased BCa cell proliferation by increasing cyclin D1 expression Through both cell-to-cell interactions and secretion of soluble factors., osteocytes increased BCa cell proliferation by increasing cyclin D1 expression. Moreover, osteocyte-derived CM stimulated BCa cells migration and invasion capabilities and upregulated the expression of genes involved in migration (Vcam-1 and N-cadherin). Co-culture with stromal cells also stimulated BCa cell proliferation, invasion, and migration. However, the increases induced by stromal cells were four-fold lower than those seen with osteocytes. Overall, our results suggest that BCa cells reprogram osteocytes, which acquire a senescent phenotype, whereas osteocytes produce factors that stimulate tumor growth, migration, and invasion. Together, these findings support the existence of a crosstalk between osteocytes and BCa cells in the metastatic niche that favors the progression of breast cancer in bone. Future studies are warranted to examine the contribution of osteocyte senescence and osteocytes-derived factors in animal models of breast cancer metastasis.

Citation Format: Manish Adhikari, Hayley Sabol, Aric anloague, Sharmin Khan, Jesus Delgado-Calle. Pathological crosstalk between osteocytes and breast cancer cells in bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5675.
Abstract 5680: Hypoxia-responsive and HIF1α-regulated AAA+ ATPase ATAD2 shows high oncogenic potential in stomach cancer
Authors: Roychowdhury Anasuya , Nayak Aditi , Kumar Sugandh , Dixit Anshuman , Bhattacharyya Asima
Published: June 2022

The burden of stomach cancer incidence and death is a global challenge. Treating stomach cancer remains immensely difficult due to the lack of available markers to detect the disease at the early stages. Therefore, there is an immense demand for finding out an early detection biomarker for stomach cancer. Recently, ATAD2 is found to be overexpressed in stomach cancer with high prognostic significance and recognised as a promising biomarker for stomach cancer. ATAD2 is a unique cancer/testis antigen (CTA) that belongs to both AAA+ ATPase and bromodomain family proteins. However, nothing much is known about the mechanism of ATAD2-mediated stomach carcinogenesis. Using bioinformatics analyses as well as studies with stomach cancer cells and stomach biopsy tissue samples, we address the regulation and function of ATAD2 expression in stomach cancer. We show that ATAD2 is overexpressed in the most common intestinal type of stomach adenocarcinoma, and enhanced expression of ATAD2 is observed in all stages and grades of stomach cancer. Our immunofluorescence microscopy study with stomach biopsy tissues confirms the high expression of ATAD2 both in stomach adenocarcinoma and metastatic stomach tissue samples. Survival analysis indicates that upregulated ATAD2 expression drastically affects the survival of stomach cancer patients. Since H. pylori infection, and hypoxia are the major contributing factors for stomach carcinogenesis; we further study their role in ATAD2-mediated stomach malignancy. An enhanced expression of ATAD2 is observed in H. pylori-infected stomach cancer cells. We identify ATAD2 as a hypoxia-responsive and HIF1α-regulated gene and elucidate that upregulated expression of ATAD2 enhances proliferation and migration of hypoxic stomach cancer cells. We further recognize the protein-protein interaction (PPI) network of ATAD2 with the top-ranked partners like ESR1 (or ERα), NCOA3 (or ACTR/SRC3/AIB1), SUMO2, HDA11 (HDAC11), SPTN2 (SPTBN2), and MYC. Among them, ESR1, MYC, NCOA3, and HDA11 are oncoproteins. Most strikingly, we report the participation of two druggable targets of ATAD2, i.e., AAA+ ATPase and bromodomain in the ATAD2-PPI network; ESR1, SUMO2, SPTN2, and MYC show a preference for bromodomain, whereas NCOA3 and HDA11 prefer ATPase domain of ATAD2. The importance of such findings in terms of targeting bromodomain-PPI and/or ATPase-PPI interfaces to curtail stomach cancer is elucidated.

Citation Format: Anasuya Roychowdhury, Aditi Nayak, Sugandh Kumar, Anshuman Dixit, Asima Bhattacharyya. Hypoxia-responsive and HIF1α-regulated AAA+ ATPase ATAD2 shows high oncogenic potential in stomach cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5680.
Abstract 5785: Tumors with higher heterogeneity were associated with poorer survival outcome in early stage lung adenocarcinoma cancer patients with low tumor mutational burden
Authors: Fridland Stanislav , Chae Young Kwang
Published: June 2022

Background: Response to immune checkpoint inhibitors is associated with tumor mutational burden (TMB). Pembrolizumab has recently been approved by the FDA for use in patients with high TMB. However low TMB patients are not considered good responders and could benefit from stratification. The predictive value of other biomarkers such as intratumor heterogeneity (ITH) has been studied across a range of cancer indications. The utility of ITH in Lung Adenocarcinoma (LUAD) has not been established. Here we evaluate the relationship between ITH and outcome in early stage low TMB patients and explore differences in immune cell enrichment and predicted driver genes. Stratifying early stage patients may yield an opportunity to improve Recurrence Free Survival (RFS) and lower the risk of progression to late stage disease.

Methods: All molecular data was obtained from the Tumor Cancer Genome Atlas via the GDC Data Portal. Clinical data was from the PanCancer Atlas dataset through cBioPortal. We estimated ITH using PyClone, an algorithm that estimates the number of tumor clones. We generated our ITH values in triplicate across 3 separate days, yielding an average PyClone clone number. Clones with >2 mutations were counted. We used p-value minimization to choose 1.4 for the TMB cutoff and 2.6 for the PyClone cutoff. We generated 4 groups: HTHI, HTLI, LTHI, and LTLI, where H - high, L- low, T-TMB, and I-ITH. Immune cell enrichment analysis was obtained from the xCell webtool. Driver gene prediction was performed with OncoDriveClust via the R package maftools.

Results: We found a statistically significant difference in RFS between early stage (Stage I and Stage II) LTHI (N = 18) and LTLI (N = 40) patients (16.69 months vs. 33.27 months, p-value = 0.0056). Interestingly the predicted driver genes did not significantly differ between these two groups. One driver (EGFR) and two drivers (EGFR and TP53) genes were predicted for LTHI and LTLI, respectively. Gene set enrichment analysis showed statistically significant differences in key immune subtypes. B-cells were depressed and Th1 cells were elevated in LTHI, both of which are associated with poorer survival outcomes.

Conclusion: We found an association between high ITH and poor survival in low TMB early stage LUAD patients. Early stage patients can be stratified using heterogeneity metrics like PyClone. Given the presence of immune subtypes known to associate with poorer outcomes, early stage LTHI patients may benefit from additional adjuvant therapies.

Citation Format: Stanislav Fridland, Young Kwang Chae. Tumors with higher heterogeneity were associated with poorer survival outcome in early stage lung adenocarcinoma cancer patients with low tumor mutational burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5785.
Abstract 5805: Novel approach to thymidylate synthase genotype determination and utility as a biomarker of severe toxicity to 5-FU chemotherapy
Authors: Huang Huixing , Schaerer Dominic , Schmidt Remington , Zhang Ting , Froehlich Tanja K , Bouchonville Kelly , Diasio Robert B , Amstutz Ursula , Largiadèr Carlo , Offer Steven M
Published: June 2022

The primary mechanism of action for the chemotherapeutic 5-fluorouracil (5-FU) is inhibition of thymidylate synthase (TS), which leads to nucleotide imbalance and subsequent cellular death. Expression of TS has been suggested to be determined by a variable number of tandem repeats located within the TS enhancer region (TSER). TSER genotype has been inconsistently linked to 5-FU-related adverse events, including severe grade ≥3 drug-related toxicity. Our recent studies demonstrated that the number of repeats within the TSER, as well as the presence or absence of a polymorphism within each repeat, was significantly associated with 5-FU toxicity in 629 patients. Our findings are consistent with a model in which the number of upstream stimulatory factor (USF1) transcription factor binding sites in the TSER, which is determined both by repeat status and genotype within each repeat, defines TYMS expression and contributions to 5-FU toxicity risk. We present an updated nomenclature that concisely and unambiguously identifies both the number of repeats and the number of USF1 binding sites. The difficulty in obtaining clear genotypes within this highly repetitive and variable region limits the ability to integrate this biomarker into predictive tests. We present methodologies for assigning TSER genotypes using conventional and high-throughput sequencing, including whole genome sequence data. Our results are expected to promote the improved study of TSER genotype in future clinical studies and the integration of TSER status into clinical predictive tests for severe 5-FU toxicity.

Citation Format: Huixing Huang, Dominic Schaerer, Remington Schmidt, Ting Zhang, Tanja K Froehlich, Kelly Bouchonville, Robert B Diasio, Ursula Amstutz, Carlo Largiadèr, Steven M Offer. Novel approach to thymidylate synthase genotype determination and utility as a biomarker of severe toxicity to 5-FU chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5805.
Abstract 5822: Leptin modulated microRNA-628-5p targets Jagged1 and inhibits prostate cancer hallmarks
Authors: Colon Leslimar Rios , Chijioke Juliet , Niture Suryakant , Afzal Zainab , Qi Qi , Srivastava Anvesha , Ramalinga Malathi , Kedir Habib , Cagle Patrice , Arthur Elena , Deep Gagan , Suy Simeng , Collins Sean , Kumar Deepak
Published: June 2022

Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer and the second leading cause of cancer deaths in men. Obesity is a major risk factor for prostate cancer. Leptin is an obesity hormone that controls body weight by regulating energy intake and expenditure, and clinical studies have demonstrated a correlation between blood leptin levels and PCa development. MicroRNAs (miRs) are master regulators of cancer cell signaling, including in advanced PCa. We previously identified miR-628-5p as a potential biomarker in PCa patients that was downregulated in serum samples of men with PCa (Tumor Biol., 2014, 35: 4867). In this study, we investigated the link between the obesity hormone leptin and the functional role of miR-628-5p in PCa cell lines. We demonstrate that leptin downregulates the expression of miR-628-5p and increases cell proliferation and migration in PCa cells. Enforced expression of miR-628-5p inhibited cell proliferation in PCa cells. Further, miR-628-5p reduced PCa cell survival/migration/invasion/spheroids formation and cell stemness. Mechanistically, miR-628-5p binds with JAG1-3’UTR and inhibits the expression of Jagged-1 (JAG1). JAG1 inhibition by miR-628-5p also downregulated Notch signaling, decreased the expression of snail/slug, and modulated epithelial-mesenchymal transition and invasiveness in PC3 cells. Furthermore, miR-628-5p increased sensitivity towards enzalutamide and docetaxel by inducing cell apoptosis. Collectively our data suggested that miR-628-5p is a key regulator of PCa carcinogenesis modulated by adipokine leptin and offers a novel therapeutic opportunity to inhibit the growth of advanced PCa.

Citation Format: Leslimar Rios Colon, Juliet Chijioke, Suryakant Niture, Zainab Afzal, Qi Qi, Anvesha Srivastava, Malathi Ramalinga, Habib Kedir, Patrice Cagle, Elena Arthur, Gagan Deep, Simeng Suy, Sean Collins, Deepak Kumar. Leptin modulated microRNA-628-5p targets Jagged1 and inhibits prostate cancer hallmarks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5822.
Abstract 588: Regional administration of IL-12 endowed CAR T cells effectively targets systemic disease
Authors: Lee Hee Jun , Priceman Saul
Published: June 2022

While chimeric antigen receptor (CAR) T cell therapy has shown impressive clinical efficacy for hematological malignancies, efficacy remains limited for solid tumors due in large part to the immunosuppressive tumor microenvironment. Tumor-associated glycoprotein 72 (TAG72) is an aberrantly glycosylated protein overexpressed on ovarian cancer and is an exciting target for CAR T cell immunotherapy. Our lab previously developed a second-generation TAG72 CAR T cell product and showed its potency against TAG72-expressing ovarian tumor cells both in vitro and in preclinical mouse models. We report here further modification of our TAG72 CAR T cells, with incorporation of interleukin-12 (IL-12) and interleukin-15 (IL-15), and evaluate the therapeutic benefits in peritoneal ovarian tumor models.

In this preclinical study, we build upon our earlier work with in vitro and in vivo evaluation of 9 different second-generation TAG72 CAR constructs varying in single-chain variable fragment, extracellular spacer, transmembrane, and intracellular co-stimulatory domains. We then engineer CAR T cells with two types of cytokines - IL-12 and IL-15 - and put these engineered cells against challenging in vivo tumor models.

Through in vitro and in vivo studies, we identified the most optimal construct with which we aim to evaluate in a phase 1 clinical trial targeting TAG72-positive ovarian cancer in early 2022. Despite thorough optimizations to the CAR backbone, CAR T cells can be additionally engineered for improved anti-tumor response. Therefore, we further engineered CAR T cells with IL-12 or IL-15 production that greatly improved the effectiveness of TAG72-CAR T cells in difficult-to-treat in vivo tumor models. We observed that modification of CAR T cells with IL-15 displayed toxicity when regionally delivered in vivo, yet introduction of IL-12 not only demonstrated safe and superior therapeutic responses, but also allowed the regional administration of CAR T cells to address systemic disease. We are now expanding these findings by evaluating these therapies using syngeneic immunocompetent mouse tumor models.

The tumor microenvironment (TME) harbors various factors that thwart the killing of tumor cells by CAR T cells. Thus, CAR T cells will likely require further engineering to overcome this barrier. We show that amplifying cytokine pathways is one way to overcome the TME and improve the efficacy of CAR T cell therapy for solid tumors.

Citation Format: Hee Jun Lee, Saul Priceman. Regional administration of IL-12 endowed CAR T cells effectively targets systemic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 588.
Abstract 5919: Regulation of SLPI secretion by NFX1-123 and HPV 16 E6 in cervical cancer
Authors: Chintala Sreenivasulu , Quist Kevin , Katzenellenbogen Rachel
Published: June 2022

Background: Secretory Leukocyte Peptidase Inhibitor (SLPI) functions in cancer progression, cellular invasion, metastasis, immune evasion, and chemoresistance. Our previous studies showed increased SLPI with overexpressed NFX1-123 and co-expressed high-risk (HR) human papillomavirus type 16 E6 oncogene (16E6) in primary keratinocytes (HFKs) as well as high SPLI levels in cervical cancers. The HR viral oncogenes E6 and E7 partner with host cell proteins to regulate cell cycle regulation and immortalization pathways. Understanding the mechanisms of SLPI upregulation in HPV-associated cancers is critical to validating SLPI as a non-invasive biomarker as well as to test its utility in future cancer treatments. This study was designed to quantify intracellular and secreted SLPI protein in cervical cancer and to determine the roles NFX1-123 and 16E6 have on SLPI expression and secretion.

Methods: Using HPV- and HPV+ cervical cancer cell lines and HFKs transduced with overexpressed NFX1-123 and 16E6, or vector controls, we measured intracellular and secreted SLPI by Western blot and ELISA, respectively. In HPV+ cervical cancer cell lines, NFX1-123 was knocked down or out by shRNA or CRISPR-Cas9, respectively, and 16E6 and 16E7 were knocked down by siRNA. In those cells, intracellular and secreted SLPI was quantified and the relative role of NFX1-123, 16E6, and 16E7 were studied. Selected classical and non-classical secretory pathways markers were also measured to assess their effects on SLPI secretion. Finally, SLPI’s concentration was measured by ELISA in cervical cancer patient sera and compared to healthy controls.

Results: Our results showed increased intracellular as well as secreted SLPI in HFKs with overexpressed NFX1-123 and 16E6. Similarly, we found increased SLPI secretion in HPV+ cervical cancer cell lines (SiHa, Caski and HeLa) when compared to HPV- C33A cells. A reduction in NFX1-123 and 16E6 led to decreased SLPI secretion, pointing to a regulation of SLPI by NFX1-123 and 16E6 on SLPI secretion. Analysis of classical and non-classical secretory pathway markers suggested that NFX1-123 regulated non-classical pathways, which may drive greater secretion of SLPI in HPV+ cervical cancers. Higher SLPI protein amounts were found cervical cancer patient sera when compared to healthy controls.

Conclusions: Our results suggest a functional role for NFX1-123 and 16E6 in SLPI secretion in cervical cancer and that SLPI may be a potential biomarker in cervical cancer.

Citation Format: Sreenivasulu Chintala, Kevin Quist, Rachel Katzenellenbogen. Regulation of SLPI secretion by NFX1-123 and HPV 16 E6 in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5919.
Abstract 5925: Albumin-bilirubin score as a marker for predicting occurrence of hepatocellular carcinoma in patients with liver cirrhosis
Authors: Toshikuni Nobuyuki , Tsuchishima Mutsumi , Tsutsumi Mikihiro , George Joseph
Published: June 2022

Background: The newly developed albumin-bilirubin (ALBI) score has been proved to be a simple and useful marker for predicting prognosis in patients with hepatocellular carcinoma (HCC). However, evidence is scarce regarding the use of ALBI score to predict occurrence of HCC in patients with chronic liver disease. In the current study, we examined the usefulness of ALBI score to predict occurrence of HCC in patients with liver cirrhosis.

Methods: In this retrospective study, we included only cirrhotic patients without previous history of HCC. A total of 68 cirrhotic patients (median age, 68 years old; 36 males and 32 females; 42 compensated and 26 decompensated cirrhosis) were included. Using a Cox proportional hazard model, we identified the risk factors for HCC occurrence and mortality. The potential factors were age, gender, causes of cirrhosis (hepatitis virus infection vs others), platelet count, serum alpha-fetoprotein (AFP) level, serum endocan level, ALBI score, and successful treatment for cirrhosis (yes vs no).

Results: The leading cause of cirrhosis was hepatitis virus infection (9 HBV- and 20 HCV-infected patients), followed by alcohol-related liver disease (23 patients), nonalcoholic steatohepatitis (5 patients), and other causes (2 patients). The median follow-up period was 10 years. During the study period, 16 patients had HCC and 22 patients died (12 liver failure, 3 HCC, 2 variceal rupture, 2 bacterial infection, and 3 other causes). Cumulative survival rates were 92%, 72%, and 59%, at 5, 10, and 15 years, respectively. Regarding risk factors for HCC occurrence, univariate analysis identified a low platelet count and an increased ALBI score as significant factors. However, on multivariate analysis, an increased ALBI score was the only significant factor [hazard ratio, 7.49 (1.65−34.04); p = 0.009]. Regarding risk factors for mortality, univariate analysis identified older age as the only significant factor; male gender and hepatitis virus infection tended to be significant. However, on multivariate analysis, older age, male gender, and hepatitis virus infection were significant factors.

Conclusion: The results indicate that an increased ALBI score is a useful factor to predict occurrence of HCC in patients with liver cirrhosis.

Citation Format: Nobuyuki Toshikuni, Mutsumi Tsuchishima, Mikihiro Tsutsumi, Joseph George. Albumin-bilirubin score as a marker for predicting occurrence of hepatocellular carcinoma in patients with liver cirrhosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5925.
Abstract 594: FcγRIIB expressed on activated CD8+ T cells restrains T cell responsiveness to αPD-1 immune checkpoint blockade in melanoma patients
Authors: Bennion Kelsey B. , Tariq Marvi , Baecher Kirsten M. , Paulos Chrystal M. , Kudchadkar Ragini , Lowe Michael C. , Ford Mandy L.
Published: June 2022

In this study, we show that FcγRIIB on the surface of CD8+ T cells reduces responsiveness to αPD-1 antibody in melanoma patients, and we suggest therapeutic strategies to improve CD8+ T cell response to PD-1 blockade. Immune checkpoint blockade (ICB) such as PD-1 blockade has drastically improved melanoma patient survival, but not all patients respond to ICB. Elevated expression of inhibitory molecules on CD8+ T cells dampens the antitumor T cell response and limits patient response to ICB. We recently showed that the inhibitory receptor FcγRIIB is expressed on effector-like memory CD8+ T cells and functions in a cell-intrinsic manner to temper activated CD8+ T cells in vivo. We found that FcγRIIB+ CD8+ T cells were more proliferative and secreted more pro-inflammatory cytokines than their FcγRIIB- counterparts. FcγRIIB+ CD8+ T cells also expressed elevated levels of exhaustion/activation markers (PD-1, CTLA-4, etc.). As these markers are also the targets of immune checkpoint blockade, we investigated the response of these cells to checkpoint therapy. Interestingly, FcγRIIB+ cells exhibited significantly reduced proliferation to αPD-1 and αCTLA-4 in vitro compared to FcγRIIB- cells (p<0.05). In stage-IV melanoma patients, we observed a decrease in FcγRIIB+ CD8+ T cells (p<0.05) following administration of nivolumab (αPD-1), a finding replicated in mouse models in vivo. Because ICB utilizes IgG antibody, a canonical ligand for FcγRs, we hypothesized that ICB antibodies could bind FcγRIIB on CD8+ T cells and elicit counterproductive negative signaling. In a B16-melanoma mouse model, genetic deletion of FcγRIIB on CD8+ T cells increased CD8+ T-cell infiltration and IFNγ production at the tumor in αPD-1 treated mice (p<0.05). Further, blocking inhibitory signaling through FcγRIIB in αPD-1-treated animals increased the number of Ki67+, TNF+, and IFNγ+ CD8+ T cells at the tumor and delayed tumor growth compared to αPD-1 treatment alone (p<0.05). Thus, we show that FcγRIIB is a novel inhibitory molecule on CD8+ T cells that limits T-cell responsiveness to αPD-1 immunotherapy and that T cell-expressed FcγRIIB should be a consideration in the development of therapeutic antibodies. We also demonstrate the efficacy of blocking FcγRIIB to enhance the CD8+ T-cell response to αPD-1 in mice, illuminating a potential new therapeutic target to improve melanoma patient response to ICB.

Citation Format: Kelsey B. Bennion, Marvi Tariq, Kirsten M. Baecher, Chrystal M. Paulos, Ragini Kudchadkar, Michael C. Lowe, Mandy L. Ford. FcγRIIB expressed on activated CD8+ T cells restrains T cell responsiveness to αPD-1 immune checkpoint blockade in melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 594.
Abstract 5948: Modeling lung squamous cell carcinoma progression using primary human bronchial basal cells
Authors: Ogden Julia , Dive Caroline , Garcia Carlos Lopez
Published: June 2022

Background: Lung squamous cell carcinoma (LUSC) arises from the accumulation of genetic driver alterations in bronchial basal cells. LUSC genomes lack obvious actionable mutations and present with a high degree of inter-tumor heterogeneity. Tractable, patient relevant models are needed to study LUSC pathogenesis and investigate the role of recurrent genomic alterations in driving cancer progression. Therefore, we are developing a tractable human model of LUSC progression to increase our understanding of this disease.

Methods: To investigate the contribution and co-operation of frequently mutated genes (TP53 and CDKN2A) and commonly deregulated pathways (SOX2/P63, PI3K/AKT and KEAP1/NRF2 pathways) in driving LUSC progression, we genetically engineered primary human bronchial basal cells. This culminated in eight basal cell mutants harboring ubiquitous LOF alterations in TP53 and CDKN2A combined with additional pathway alterations. To assess the function of the genetic alterations in LUSC progression, we investigated proliferation using cell cycle analysis and colony forming assays. Air-liquid interface organotypic cultures were used to model histological changes in the bronchial epithelium driven by different combinations of mutations. The resultant cultures were analyzed for changes in histology indicative of LUSC progression using immunostaining to evaluate changes to epithelial cell proliferation, and expression of LUSC biomarkers.

Results: Increases in proliferation were driven by inactivation of TP53, CDKN2A and PTEN, whereas SOX2 overexpression inhibited mutant basal cell proliferation. LUSC alterations also drove striking phenotypic changes in 3D organotypic cultures of the bronchial epithelium. All mutant cultures displayed thickening of the bronchial epithelium and significantly increased cellularity (p<0.001), indicating hyper-proliferation. SOX2 overexpression inhibited mucociliary differentiation and drove the appearance of a disordered epithelium with clear cellular and nuclear pleomorphism, indicative of high-grade premalignant lesions.

Conclusions: We developed a basal cell model of recurrent LUSC genetic alterations. This approach is unique due to [1] the use of primary human cells and [2] the representation of interpatient heterogeneity and developmental stages by introducing different combinations of pathway alterations. Early results indicate that basal cells carrying cumulative alterations display increasingly complex phenotypes with features of low and high-grade premalignant lesions. Further work is required to assess the tumorigenicity of mutant cells.

Citation Format: Julia Ogden, Caroline Dive, Carlos Lopez Garcia. Modeling lung squamous cell carcinoma progression using primary human bronchial basal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5948.
Abstract 5950: A novel biguanide as a potential head and neck cancer therapeutic in Fanconi anemia
Authors: Archibald Hunter , Wuertz Beverly , Ondrey Frank
Published: June 2022

Introduction: Fanconi Anemia (FA) patients develop head and neck squamous cell carcinoma (HNSCC) at significantly elevated rates and conventional adjunctive chemotherapy and radiation are exquisitely toxic to these patients. We endeavor to find a safer chemotherapeutic agent for treatment of head and neck cancer in this population. Currently, agents such as metformin are in FA clinical trials for marrow failure and are also high interest agents in head and neck cancer chemoprevention. HNSCC in FA is a disease model that has applications to typical head and neck cancer, as its mutational defects are felt to be similar.

Methods: We completed cell proliferation assays on FA-derived HNSCC cell lines MOP FA-1 and VU-1131. MTT, Cell Titer Glo, trypan blue, and clonogenic assays were completed on each cell line to evaluate the effect of N1-hexyl-N5-benzyl-biguanide (HBB) in comparison with standard cisplatin chemotherapy. We also compared the caspase activity in an effort to evaluate a putative mechanism of HBB cellular inhibition.

Results: HBB showed a statistically significant dose-dependent inhibition of cellular proliferation. In MTT assays, 5 uM HBB showed 28-46% inhibition at 72 hours and 10uM HBB showed 77-82% inhibition at 72 hours. There was an additive inhibition of cellular proliferation when HBB was combined with low-dose cisplatin.

Conclusion: HBB is a novel biguanide that inhibited cellular proliferation in two FA HNSCC cell lines and chemosensitized to low-dose cisplatin. This may be an alternative adjunct treatment agent, or may be chemosensitizing for low-dose cytotoxic agents such as platinum-based chemotherapeutics in this unique population.

Citation Format: Hunter Archibald, Beverly Wuertz, Frank Ondrey. A novel biguanide as a potential head and neck cancer therapeutic in Fanconi anemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5950.
Abstract 5968: Ethnodyne Visio™ affects EGFR signaling in breast cancer
Authors: Samant Rishab Rajeev , Samant Rajeev
Published: June 2022

Breast Cancer (BC), the most common form of female cancer, is one of the most extensively studied diseases. Metastasis is the process by which a cancer cell travels to another part of the body and starts to multiply. This detrimental occurrence is one of the primary forms of disease relapse in cancer patients and accounts for an estimated 90 percent of cancer deaths. Ethnodyne Visio™ is a new drug for Age-Related Macular Degeneration (AMD), the primary cause for blindness in people 65 and older. It is a mixture of phytochemicals Withaferin A, Gallic Acid, and Bacopaside I. However, the exact mechanism of action of Ethnodyne Visio™ is not known. Macular Degeneration occurs in two primary forms: wet and dry. Wet Macular Degeneration shows surprising similarities with metastatic Breast Cancer in terms of their progression and causation. As both diseases evolve, the retina (in the case of Wet AMD) and the tumor (in the case of BC) secrete Vascular Endothelial Growth Factor (VEGF) to encourage vascular development. In Wet AMD, this new and unregulated vascular growth, or angiogenesis, causes “leaky” blood vessels that allow fluid to enter the retina, ultimately causing blindness. In BC, this blood vessel growth provides a means for the tumor to obtain nutrients as well as disseminate metastatic cells. We determined if there exists any easily accessible nutraceuticals possessing effective antiangiogenic properties. We used CAMs (chick chorioallantoic membrane), grown ex-ovo, to study angiogenesis. We found that Ashwagandha (Withaferin A) blocked angiogenesis of CAMs by 67%. Taking clues from these observations, we wanted to determine possible molecular signaling mechanisms triggered by Ethnodyne Visio™ that may counter progression of BC. We hypothesized that Ethnodyne Visio™ may have anti-angiogenic properties. Using multiple online gene association platforms, we compiled a list of transcriptomic changes caused by Withaferin A, Gallic Acid, and Bacopaside I on breast tumor cells. A g:Profiler analysis of the combined transcriptomic changes revealed Epidermal Growth Factor Receptor (EGFR) signaling as a downstream target of the components of Ethnodyne Visio™. Our analysis reveals that Ethnodyne Visio™ potentially regulates VEGF signaling through the EGFR pathway.

Citation Format: Rishab Rajeev Samant, Rajeev Samant. Ethnodyne Visio™ affects EGFR signaling in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5968.
Abstract 5971: DEPDC1B promotes melanoma angiogenesis and metastasis through sequestration of ubiquitin ligase CDC16 to stabilize secreted SCUBE3
Authors: Hu Feng , Fong Ki On , Cheung May P.L , Liu Jessica A.J , Liang Rui , Li Tsz Wai , Sharma Rakesh , Ip Philip P.C , Yang Xintao , Cheung Martin
Published: June 2022

The ability of melanoma to acquire metastasis through the induction of angiogenesis is one of the major causes of skin cancer death. Here, we find that high transcript levels of DEPDC1B in cutaneous melanomas are significantly associated with a poor prognosis. Tissue microarray analysis indicates DEPDC1B expression is positively correlated with SOX10 in the different stages of melanoma. Consistently, DEPDC1B is both required and sufficient for melanoma growth, metastasis, angiogenesis, and functions as a direct downstream target of SOX10 to partly mediate its oncogenic activity. In contrast to other tumor types, the DEPDC1B-mediated enhancement of melanoma metastatic potential is not dependent on the activities of RHO GTPase signaling and canonical Wnt signaling, but is acquired through secretion of SCUBE3, which is crucial for promoting angiogenesis in vitro and in vivo. Mechanistically, DEPDC1B regulates SCUBE3 protein stability through the competitive association with ubiquitin ligase CDC16 to prevent SCUBE3 from undergoing degradation via the ubiquitin-proteasome pathway. Importantly, expression of SOX10, DEPDC1B, and SCUBE3 are positively correlated with microvessel density in the advanced stage of melanomas. In conclusion, we reveal a SOX10-DEPDC1B-SCUBE3 regulatory axis promotes melanoma angiogenesis and metastasis, which suggests targeting secreted SCUBE3 can be a therapeutic strategy against metastatic melanoma.

Citation Format: Feng Hu, Ki On Fong, May P.L Cheung, Jessica A.J Liu, Rui Liang, Tsz Wai Li, Rakesh Sharma, Philip P.C Ip, Xintao Yang, Martin Cheung. DEPDC1B promotes melanoma angiogenesis and metastasis through sequestration of ubiquitin ligase CDC16 to stabilize secreted SCUBE3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5971.