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Knockdown of GH receptor (GHR) in melanoma cells in vitro downregulates ATP-binding cassette-containing (ABC) transporters and sensitizes them to anti-cancer drug treatments. Here we aimed to determine whether a GHR antagonist (GHRA) could control cancer growth by sensitizing tumors to therapy through downregulation of ABC transporters in vivo. We intradermally inoculated Fluc-B16-F10 mouse melanoma cells into GHA mice, transgenic for a GHR antagonist (GHRA), and observed a marked reduction in tumor size, mass and tumoral GH signaling. Moreover, constitutive GHRA production in the transgenic mice significantly improved the response to cisplatin treatment by suppressing expression of multiple ABC transporters and sensitizing the tumors to the drug. We confirmed that presence of a GHRA and not a mere absence of GH is essential for this chemo-sensitizing effect using Fluc-B16-F10 allografts in GH knockout (GHKO) mice, where tumor growth was reduced relative to that in GH-sufficient controls but did not sensitize the tumor to cisplatin. We extended our investigation to hepatocellular carcinoma (HCC) using human HCC cells in vitro and a syngeneic mouse model of HCC with Hepa1-6 allografts in GHA mice. Gene expression analyses and drug-efflux assays confirm that blocking GH significantly suppresses the levels of ABC transporters and improves the efficacy of sorafenib towards almost complete tumor clearance. Human patient data for melanoma and HCC show that GHR RNA levels correlate with ABC transporter expression. Collectively, our results validate in vivo that combination of a GHRA with currently available anti-cancer therapies can be effective in attacking cancer drug resistance.

Background: Oncogenic mutations in KRAS are expressed in up to 90% of pancreatic ductal adenocarcinomas (PDAC). Vaccination against mutant KRAS (mKRAS) is thus a promising approach as an off-the-shelf immunotherapeutic treatment for PDAC. We developed a mKRAS peptide vaccine targeting 6 common KRAS mutations (G12V, G12A, G12C, G12R, G12D, or G13D (NCT04117087). We evaluated the mKRAS specific T cell response induced by vaccination in combination with immune checkpoint inhibitors (ICIs) in patients with resected PDAC. Materials and Methods: This is an ongoing pilot study of a pooled mKRAS long peptide vaccine in combination with ICIs in patients with resected PDAC and mutations in one of 6 KRAS mutations in our vaccine. Patients with no evidence of disease on imaging within 6 months of completion of adjuvant chemotherapy were vaccinated with the mKRAS vaccine (0.3mg/peptide and 0.5mg poly-ICLC (Hiltonol: Oncovir)) weekly for 4 doses followed by booster vaccines every 8 weeks. Patients also received ipilimumab (1mg/kg, every 6 weeks for 2 doses) and nivolumab (3mg/kg, every 3 weeks in the priming phase) followed by nivolumab (480mg, flat dose in boost phase). To evaluate the expansion of mKRAS-specific T cells in the periphery, pre- and post-vaccination peripheral blood mononuclear cells (PBMCs) were restimulated with control or individual mKRAS peptides and assessed for IFNγ release by ELISPOT. To further phenotype the responding CD4 and CD8 T cell compartments, peptide-restimulated PBMCs were evaluated for T cell activation, proliferation, memory and exhaustion marker expression by CyTOF. Paired single-cell RNA and TCR sequencing are being performed to isolate mKRAS-specific TCRs and their corresponding transcriptional profiles. These TCRs are being functionally validated in vitro using CRISPR-Cas12a-based genome knock-in of human T cells. Results: At the time of data cut off (1/12/2023) 8/11 patients enrolled had a positive mKRAS-specific T cell response in post-vaccine sample timepoints defined by a >5 fold increase in IFNγ producing T cells after peptide restimulation. CyTOF analysis of peptide-restimulated PBMCs demonstrated expansion of polyfunctional (IFNγ+IL2+TNFα+) mKRAS-specific CD4 and CD8 T cells with both central and effector memory phenotypes after vaccination. mKRAS-specific CD4 T cells were induced at a greater proportion. Our single-cell analysis has identified and validated a novel CD4+ TCR that recognizes KRAS G12V in the context of HLA-DRB1*07:01. Conclusions: This study thus far indicates the induction of de novo, high quality mKRAS-specific T cells in the periphery post-vaccine. Ongoing studies will define TCR diversity and clonality of mKRAS-specific T cells to each mKRAS epitope. Overall, our data will be used to identify peripheral T cell-based biomarkers that may be able to predict response to mKRAS-targeted immunotherapy. Citation Format: Amanda L. Huff, Saurav D. Haldar, Emily Davis-Marcisak, Thatcher Heumann, Gabriella Longway, Alexei Hernandez, Maximillian F. Konig, Brian Mog, Ludmila Danilova, Luciane Kagohara, Julie M. Nauroth, Amy M. Thomas, Elana J. Fertig, Won Jin Ho, Elizabeth M. Jaffee, Nilo Azad, Neeha Zaidi. A pooled mutant KRAS peptide vaccine activates polyfunctional T cell responses in patients with resected pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB197.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a low tumor mutational burden and therefore few neoantigen targets that can be recognized by cytotoxic T cells. Most PDACs are thus insensitive to either single or dual immune checkpoint inhibitor (ICI) therapy. Personalized neoantigen vaccines can expand the number and repertoire of anti-tumor T cells that infiltrate the tumor and mediate cytotoxicity. To model a personalized neoantigen vaccine treatment strategy in PDAC, we previously developed PancVAX, a peptide-based vaccine targeting 12 neoantigens expressed in the murine pancreatic cell line Panc02 (Kinkead et al, JCI Insight 2018). Although we observed increased T cell infiltration present in the tumor post-vaccination, these cells expressed high levels of exhaustion markers. We therefore hypothesized that sequential administration of anti-CTLA-4 and anti-PD-1 would enhance the pool of T cells primed by the neoantigen vaccine and maintain activation of antigen-experienced T cells, respectively, to yield optimal and durable neoantigen-specific anti-tumor immunity in PDAC. To address this, mice bearing subcutaneous Panc02 tumors were vaccinated with two rounds of the PancVAX neoantigen vaccine followed by anti-CTLA-4 and anti-PD-1 3 days later. Anti-PD-1 maintenance was given twice weekly beginning at the first vaccine dose. Twelve days after the last peptide vaccine dose, tumors were harvested and dissociated into single-cell suspensions for paired single-cell RNA-sequencing and TCR-sequencing. Mice that were untreated or given ICIs without PancVAX had the highest proportions of CD8+ T cells expressing exhaustion markers. PancVAX-treated mice had more intratumoral cycling CD8 T cells and effector CD8+ T cells with high cytotoxic gene expression. Among mice treated with PancVAX, tumors from mice treated with PancVAX + anti-PD1 or PancVAX + anti-PD1 + anti-CTLA-4 had the highest proportions of effector CD8+ T cells. Ongoing analyses include differential gene expression and pathway analysis between treatment conditions in the T cell compartment in mice treated with combination ICI and PancVAX. Additionally, we will assess changes in T cell clonality and diversity within the tumors when mice are treated with single or combination therapy. These results will define a transcriptional signature associated with the generation of a productive anti-tumor immune response when neoantigen vaccines and ICI are used in combination. This work demonstrates how the addition of ICIs to personalized neo-epitope vaccines for PDAC can further enhance the quality of vaccine-induced T cell effector function in an otherwise immunologically cold tumor type and supports their inclusion in neoantigen vaccination strategies for patients with PDAC. Citation Format: Jacob T. Mitchell, Amanda Huff, Emily Davis-Marcisak, Fangluo Chen, Todd D. Armstrong, Luciane T. Kagohara, James Leatherman, Rulin Wang, Srinivasan Yegnasubramanian, Elizabeth M. Jaffee, Elana J. Fertig, Neeha Zaidi. Combination PancVAX neo-epitope vaccine with anti-CTLA-4 and anti-PD-1 antibodies enhances infiltration of cytotoxic T cells and mitigates T cell exhaustion in a murine model of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5076.

Pancreatic cancer is responsible for more than 48,000 deaths and 60,000 new cancer cases every year in the United States while the overall 5-year survival rate is only 10%. These numbers point towards an urgent need for new therapeutic approaches wherein therapy resistance poses a tremendous challenge. We and others have identified the role of growth hormone (GH) and its cognate receptor (GHR), overexpressed in pancreatic ductal adenocarcinoma patients (PDAC; >90% of pancreatic cancer cases), in driving therapy resistance by directly upregulating multidrug efflux, phenotype switch via epithelial-to-mesenchymal transition (EMT) and inhibition of apoptosis. Our laboratory had pioneered the discovery of pharmacologic GHR antagonists including the only approved and prescribed GHR antagonist, pegvisomant, and a new antagonist candidate named compound-G. Here, we used in vitro, in vivo and in silico analyses to assess the feasibility and efficacy of a treatment regimen including GHR antagonist and chemotherapy to manage PDAC. Both pegvisomant and compound-G exhibit significant suppression of cancer cellular viability, invasive potential, drug efflux rate and apoptosis in PDAC cell cultures. In male and female Nude mice with human PDAC xenografts, GHR inhibition by either pegvisomant or compound-G markedly re-sensitized the tumors towards both low and high doses of gemcitabine treatment. Compound G plus high doses of gemcitabine lead to almost complete tumor clearance in 40% of the mice. Also, tissue analyses reveal increased production of GH in tumors treated with chemotherapy. In the GHR antagonist treated tumors, significantly lower expression of ABC transporter (Abcb1, Abcg5, Abcc2, Abcg2), EMT mediator (Cdh2, Zeb1, Snai2, Epas1) and apoptosis marker (Bcl2) genes compared to control were observed - accounting for the observed sensitization to gemcitabine. In the human PDAC patient datasets, increased expression of GHR correlates strongly with increased expression of multidrug efflux transporters, anti-apoptotic markers, and EMT transcription factors. Therefore, we provide robust pre-clinical validation for a treatment regimen combining GHR antagonist and chemotherapy leading to highly efficacious tumor clearance. Acknowledgement: This work was supported in part by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by the AMVETS, and Ohio University’s Student Enhancement Award and Edison Biotechnology Institute. Citation Format: Reetobrata Basu, Prateek Kulkarni, Emily Davis, Silvana Duran, John J. Kopchick. Combination of growth hormone receptor antagonist and gemcitabine leads to highly efficacious tumor clearance in a mouse model of pancreatic ductal adenocarcinoma [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 2620.

Knockdown of the growth hormone receptor (GHR) in melanoma cells downregulates ATP-binding cassette (ABC) transporters and sensitizes them to multiple drug treatments in vitro. The goal for this study is to understand whether a GHR antagonist (GHA) could suppress different types of cancers by sensitizing tumors to drug treatments in vivo through the downregulation of ABC transporters. Sera from GHA transgenic mice inhibited the proliferation of mouse melanoma cells (B16F10) in culture and suppressed expression of multiple ABC transporters. When B16-F10 cells were intradermally inoculated into GHA mice, tumor size was markedly reduced, as was STAT5 activation and ABCG1, ABCG2 levels. We then tested the effect of the GHA on the efficacy of cisplatin in vivo. GHA sensitized melanomas to cisplatin, leading to the smallest tumors among all groups. GHKO mice showed the same effect. We further extended this investigation to hepatocellular carcinoma (HCC). GHA mice were subcutaneously inoculated with mouse HCC (Hepa1 6 cells) and subsequently treated with sorafenib. The HCC tumors in GHA mice were markedly sensitive to sorafenib treatment compared to the same in wild-type mice. RNA analysis showed that when HCC was exposed to the combined treatment, relatively decreased ABC transporters expression was found. Immunohistochemical staining showed that phosphorylation of STAT5 and ABCB1 were downregulated in HCC tissues, suggesting that GHA in vivo downregulates ABC transporters in HCC, and therefore sensitizes them to sorafenib. Clinical data derived from HCC patients using the TCGA database showed that multiple ABC transporters in both types of cancer correlate with GHR levels; that is, when GHR levels are relatively high, patient survival is significantly decreased. Additionally, higher ABCC1 levels also lead to significantly decreased survival rate in HCC patients. Collectively, the results indicate that a GHA is effective in sensitizing both melanoma and HCC to available treatments in vivo and may be used as a therapeutic strategy for higher efficacy of tumor clearance. Citation Format: Yanrong Qian, Reetobrata Basu, Samuel Mathes, Prateek Kulkarni, Emily Davis, Darlene Berryman, Edward List, Silvana Duran, John J. Kopchick. Growth hormone receptor antagonist sensitizes melanoma and hepatocarcinoma to drug treatments via downregulation of ABC transporters in vivo [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 2621.

Growth hormone (GH) has been reported to drive leukemia cell proliferation as early as 1977, while downstream effectors of the GH signaling pathway (i.e., insulin-like growth factor 1; IGF1) have more recently been widely implicated in the aggressive pathology and robust therapy resistance phenotypes of acute myeloid leukemia (AML). It is also known that patients with acromegaly (high levels of circulating GH and IGF1) develop AML at a significantly higher rate than the general population, while individuals with GH resistance remain completely resistant to all types of cancer. Despite all of the aforementioned evidence pointing to a critical role of GH in AML progression and prognosis, a detailed transcriptomic analysis of human patients is lacking. We aim to address this gap by thorough in silico studies aimed at revealing the nature and extent to which the GH axis can be suitable targeted in AML. Here, we present a summary of our analyses of clinical and transcriptomic data from 200 human patients with AML from The Cancer Genome Atlas database, using multiple bioinformatics software programs in the public domain. Our study revealed that GH receptor (GHR) is expressed more highly in AML than in normal myeloid cells, and a robust correlation exists between GH responsiveness and patient survival. A sex-specific profiling of the patient transcriptomic data identified critical pathways related to therapy resistance, extracellular matrix remodeling, metastasis, and immune evasion to be upregulated differentially in male and female AML patients with relatively elevated GHR expression compared to patients with relatively lower GHR expression. Furthermore, we identified microRNAs which are differentially expressed in AML patients with higher than median GHR expression. We validated these miRNAs to be effective indicators of disease progression and prognosis and can be valuable markers in liquid biopsy approaches. Acknowledgement: This work was supported in part by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by the AMVETS, and Ohio University’s Edison Biotechnology Institute. Citation Format: Emily Davis, Reetobrata Basu, John J. Kopchick. Transcriptomic profiling of human patients with acute myeloid leukemia reveals critical effects of growth hormone responsiveness on patient survival and mechanisms of tumor therapy resistance [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 2695.

Data from cell culture, mice, and human epidemiological studies have identified and validated growth hormone receptor (GHR) expression and growth hormone (GH) action as an oncogenic factor in cancer. Recent studies by us and others have revealed a unique aspect of GH mediated endocrine regulation of pharmacological therapy resistance in human melanoma and breast cancer. GH overexpression was found to not only promote tumor proliferation, migration, and invasion but also associated with refractoriness towards radiation and chemotherapy. However, the underlying mechanisms of the observed GH effects were unknown. To understand the molecular details of therapy resistance of the GH-GHR axis in cancer, we have used four different GHR-positive human cancers of pancreas, kidney, lung, and melanoma. We have identified multi-modal therapy resistance pathways regulated by GH action in these tumors, which was absent in GHR-negative cell lines. Here we report our latest findings explaining the method of action of GH driven cancer therapy resistance and the associated mediators. Expression of GHR and an autocrine / paracrine GH production, elicited specifically by onset of therapy (chemo, targeted, radiation) upregulates (i) ATP-binding cassette containing multidrug efflux pumps, (ii) phenotype switch via epithelial-to-mesenchymal transition, (iii) extracellular matrix rearrangement to promote tumor invasion, and (iv) cytokine mediated immunoediting. Attenuation of the GHR activity using GHR-antagonists or CRISPR-Cas9 mediated GHR knockout severely sensitized the cells towards therapy. In vivo studies using syngeneic mouse models and a thorough analyses of the TCGA data provide robust confirmation to our in vitro findings. Based on the above and ongoing studies in different mouse models of GH action, we speculate that targeting GHR as an adjuvant in cancer therapeutics might synergize therapeutic applications and lead towards a significantly improved prognosis for GHR-positive cancers.

Growth hormone (GH) and the GH receptor (GHR) are expressed in a wide range of malignant tumors including melanoma. However, the effect of GH/insulin-like growth factor (IGF) on melanoma in vivo has not yet been elucidated. Here we assessed the physical and molecular effects of GH on mouse melanoma B16-F10 and human melanoma SK-MEL-30 cells in vitro. We then corroborated these observations with syngeneic B16-F10 tumors in two mouse lines with different levels of GH/IGF: bovine GH transgenic mice (bGH, high GH, high IGF-1) and GHR gene-disrupted or knockout mice (GHRKO, high GH, low IGF-1). In vitro, GH treatment enhanced mouse and human melanoma cell growth, drug retention and cell invasion. While the in vivo tumor size was unaffected in both bGH and GHRKO mouse lines, multiple drug-efflux pumps were up regulated. This intrinsic capacity of therapy resistance appears to be GH dependent. Additionally, epithelial-to-mesenchymal transition (EMT) gene transcription markers were significantly upregulated in vivo supporting our current and recent in vitro observations. These syngeneic mouse melanoma models of differential GH/IGF action can be valuable tools in screening for therapeutic options where lowering GH/IGF-1 action is important.

Background: Immune checkpoint inhibitors (ICIs) provide benefit for immunogenic cancers that naturally attract T cell infiltration, but have limited benefit for patients with tumors that lack natural T cells in the tumor microenvironment (TME) including many breast cancers. Suboptimal and inconsistent immune responsiveness in many cancers is likely the result of a lack of tumor antigen expression and/or recognition together with multiple suppressive signals within the TME that provide a formidable barrier to T cell infiltration. Emerging data suggest that these factors can be overcome by combining epigenetic modulation that reprograms myeloid-derived suppressor cells (MDSCs) and upregulate T cell-attracting signals within the TME, with ICIs. Our paralleled preclinical and clinical trial investigates the mechanisms and efficacy of the epigenetic modulator, entinostat (ENT) to reprogram the immune suppressive cells within the TME. Methods: We conducted a multicenter phase 1 clinical trial (NCT02453620) of entinostat combined with nivolumab +/- ipilimumab in patients with advanced cancers including hormone-receptor positive or triple-negative breast cancer (TNBC). Mandatory tumor samples are being obtained pre-treatment, post-treatment with 2 weeks of entinostat, and 8 weeks post combination therapy with ICIs. We used immunohistochemical (IHC) for CD8 and FoxP3 to determine a ratio representative of immune infiltration in response to therapy. Preclinical studies utilize mouse models of breast cancer (NeuN and 4T1) and mirror the treatment scheme used in the clinical trial. We employed multiparameter flow cytometry, single cell RNA sequencing, bulk RNA sequencing of tumor specimens and patient samples to specifically interrogate the role of ENT in modulating STAT3 as a master regulator of downstream inflammatory pathways. Results: With regard to the clinical trial, out of 35 patients enrolled, 11 were diagnosed with breast cancer. We observed 4 partial responses, 2 responses in patients with breast cancer, with an ORR of 12%. We have almost completed accrual of our expansion cohort of 15 patients with advanced HER2 negative breast cancer. The primary outcomes of the clinical trial will be reported elsewhere, here we are focusing on important correlative findings which suggest a combination of ENT with ICIs alters CD8/FoxP3 ratios in certain patients. Bulk-RNA sequencing or patient samples is underway. Preliminary data from animal models obtained using scRNA-seq has begun to elucidate altered MDSC signaling pathways, and to identify gene expression changes in immune, stromal, and tumor cells following treatment with ENT. These data are also expected to identify new targets for ICI sensitization. Conclusions: Preliminary results with IHC staining suggests increased immune infiltration with combination therapy. Sequencing analysis will likely provide deeper insight into mechanisms driving response. The mechanisms of response to ICIs in patients with breast cancer have yet to be elucidated. These studies will provide the necessary scientific evidence to uncover mechanisms behind the transformation of the immunosuppressive TME and provide new targets that could improve clinical response to ICIs to maximize patient survival. The core biological themes explored will likely have a broad impact in the field of breast cancer and affect the paradigm of therapies available to patients in the clinic. These findings will help determine how treatment with ICIs will be successful to obtain durable responses for breast cancer patients. Citation Format: Evanthia T Roussos Torres, Luciane Tsukamoto Kagohara, Emily Davis, Christine Rafie, Brian Christmas, Qingfeng Zhu, Chenguang Wang, David Lim, Robert Anders, Elana Fertig, Vincent Chung, Patricia Lorusso, Adam Brufsky, Elizabeth M Jaffee, Vered Stearns, Roisin Connolly. Reprogramming the suppressive tumor microenvironment of breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-06.

CFTR modulators have revolutionized the treatment of individuals with cystic fibrosis (CF) by improving the function of existing protein. Unfortunately, almost half of the disease-causing variants in CFTR are predicted to introduce premature termination codons (PTC) thereby causing absence of full-length CFTR protein. We hypothesized that a subset of nonsense and frameshift variants in CFTR allow expression of truncated protein that might respond to FDA-approved CFTR modulators. To address this concept, we selected 26 PTC-generating variants from four regions of CFTR and determined their consequences on CFTR mRNA, protein and function using intron-containing minigenes expressed in 3 cell lines (HEK293, MDCK and CFBE41o-) and patient-derived conditionally reprogrammed primary nasal epithelial cells. The PTC-generating variants fell into five groups based on RNA and protein effects. Group A (reduced mRNA, immature (core glycosylated) protein, function 1% (n = 5)), Group D (reduced mRNA, mature protein, function >1% (n = 5)) and Group E (aberrant RNA splicing, mature protein, function > 1% (n = 1)) variants responded to modulators. Increasing mRNA level by inhibition of NMD led to a significant amplification of modulator effect upon a Group D variant while response of a Group A variant was unaltered. Our work shows that PTC-generating variants should not be generalized as genetic ‘nulls’ as some may allow generation of protein that can be targeted to achieve clinical benefit., Author summary The development of variant specific modulators that correct dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) protein is an excellent example of precision medicine. Currently there is no molecular treatment available for individuals with cystic fibrosis (CF) carrying nonsense or frameshift variants because such variants introduce a premature termination codon (PTC), and are not expected to produce CFTR protein. We have performed a systematic study of nonsense and frameshift variants located in four regions of CFTR that we postulated should have varying effects on mRNA stability, protein production, and/or function. Using primary nasal cells and three different cell line models stably expressing CFTR expression mini-genes (EMGs), we report molecular consequences of 26 PTC-generating variants in CFTR, and identify which variants allow generation of CFTR protein that is responsive to currently available modulator therapies and which require alternative therapeutic approaches.

Treatment of individuals with cystic fibrosis (CF) has been transformed by small molecule therapies that target select pathogenic variants in the CF transmembrane conductance regulator (CFTR). To expand treatment eligibility, we stably expressed 43 rare missense CFTR variants associated with moderate CF from a single site in the genome of human CF bronchial epithelial (CFBE41o-) cells. The magnitude of drug response was highly correlated with residual CFTR function for the potentiator ivacaftor, the corrector lumacaftor, and ivacaftor-lumacaftor combination therapy. Response of a second set of 16 variants expressed stably in Fischer rat thyroid (FRT) cells showed nearly identical correlations. Subsets of variants were identified that demonstrated statistically significantly higher responses to specific treatments. Furthermore, nearly all variants studied in CFBE cells (40 of 43) and FRT cells (13 of 16) demonstrated greater response to ivacaftor-lumacaftor combination therapy than either modulator alone. Together, these variants represent 87% of individuals in the CFTR2 database with at least 1 missense variant. Thus, our results indicate that most individuals with CF carrying missense variants are (a) likely to respond modestly to currently available modulator therapy, while a small fraction will have pronounced responses, and (b) likely to derive the greatest benefit from combination therapy.

Growth hormone (GH) facilitates therapy resistance in the cancers of breast, colon, endometrium, and melanoma. The GH-stimulated pathways responsible for this resistance were identified as suppression of apoptosis, induction of epithelial-to-mesenchymal transition (EMT), and upregulated drug efflux by increased expression of ATP-binding cassette containing multidrug efflux pumps (ABC-transporters). In extremely drug-resistant melanoma, ABC-transporters have also been reported to mediate drug sequestration in intracellular melanosomes, thereby reducing drug efficacy. Melanocyte-inducing transcription factor (MITF) is the master regulator of melanocyte and melanoma cell fate as well as the melanosomal machinery. MITF targets such as the oncogene MET, as well as MITF-mediated processes such as resistance to radiation therapy, are both known to be upregulated by GH. Therefore, we chose to query the direct effects of GH on MITF expression and activity towards conferring chemoresistance in melanoma. Here, we demonstrate that GH significantly upregulates MITF as well as the MITF target genes following treatment with multiple anticancer drug treatments such as chemotherapy, BRAF-inhibitors, as well as tyrosine-kinase inhibitors. GH action also upregulated MITF-regulated processes such as melanogenesis and tyrosinase activity. Significant elevation in MITF and MITF target gene expression was also observed in mouse B16F10 melanoma cells and xenografts in bovine GH transgenic (bGH) mice compared to wild-type littermates. Through pathway inhibitor analysis we identified that both the JAK2-STAT5 and SRC activities were critical for the observed effects. Additionally, a retrospective analysis of gene expression data from GTEx, NCI60, CCLE, and TCGA databases corroborated our observed correlation of MITF function and GH action. Therefore, we present in vitro, in vivo, and in silico evidence which strongly implicates the GH&ndash, GHR axis in inducing chemoresistance in human melanoma by driving MITF-regulated and ABC-transporter-mediated drug clearance pathways.

The purpose of this study is to identify how the epigenetic modulator entinostat reprograms myeloid derived suppressor cells (MDSCs) to prime the tumor microenvironment (TME) and modulate immunotherapy response. Early trials investigating single agent ICIs, anti-CTLA-4 and anti-PD-1/PD-L1, resulted in overall response rates of 5-20% in patients with metastatic HER2 positive breast cancer but little is understood about use of these therapies in patients with early stage/ primary disease. Suboptimal and inconsistent immune responsiveness is likely the result of multiple suppressive signals within the TME that provide a formidable barrier to T cell infiltration leading to an opportunity for TME modulation to potentially improve response. We and others have shown that immunosuppressive factors can be overcome by adding the class 1 histone deacetylase inhibitor, entinostat, to ICIs to reprogram MDSCs and upregulate T cell attracting signals within the TME. We hypothesize that gene expression signatures of reprogrammed MDSCs are predictive of response with subsequent immunotherapy treatment. To test this hypothesis, we used the neu-N syngeneic mouse model of HER2 positive breast cancer. We investigated differences in survival and tumor burden in response to combinations of entinostat with anti-PD-1 and anti-CTLA-4. Having previously identified MDSCs as important targets of entinostat that modulate response to ICIs, we used methods we developed to study MDSC function. Given the broad effects of entinostat on global gene expression including that of MDSCs, we used single cell RNA-seq (scRNA-seq) on primary tumors and isolated intratumoral MDSCs to determine what is driving changes in MDSC function in primary tumors. We plan to use data obtained from scRNA-seq to develop signatures of response and identify changes in responders vs. non-responders to help develop biomarkers of response in primary disease. Our results thus far show that treatment with entinostat + ICIs improves survival, and decreases tumor burden in primary tumors. Ex-vivo studies of MDSCs demonstrate changes in immunosuppressive capabilities of MDSCs and show differences in infiltration and function of granulocytic vs. monocytic MDSCs. We expect that evaluation of the ongoing sequencing data will identify more specific immune targets within primary TME and help us understand the importance of targeting MDSCs in early disease. We predict this work will identify additional candidate targets for therapeutic testing to convert breast cancers into immune responsive tumors, and that this will facilitate improved response to ICIs that will ultimately translate into improved survival of patients with breast cancer. Citation Format: Evanthia T. Roussos Torres, Christine Rafie, Emily Davis, Luciane T. Kagohara, Elana J. Fertig, Elizabeth M. Jaffee. Sequencing the tumor microenvironment and myeloid derived suppressor cells to understand response to immunotherapy in primary HER2 positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5027.

T-box factors play critical roles in embryonic development and have been implicated in cell cycle regulation and cancer. For example, Tbx2 can suppress senescence through a mechanism involving the repression of the cyclin-dependent kinase inhibitors, p19(ARF) and p21(WAF1/CIP1/SDII), and the Tbx2 gene is deregulated in melanoma, breast and pancreatic cancers. In this study, several transformed human lung fibroblast cell lines were shown to downregulate Tbx2. To further investigate the role of Tbx2 in oncogenesis we therefore stably reexpressed Tbx2 in one such cell line. Compared to their parental cells, the resulting Tbx2-expressing cells are larger, with binucleate and lobular nuclei containing double the number of chromosomes. Moreover, these cells had an increase in frequency of several features of genomic instability such as chromosome missegregation, chromosomal rearrangements and polyploidy. While grossly abnormal, these cells still divide and give rise to cells that are resistant to the chemotherapeutic drug cisplatin. Furthermore, this is shown to be neither species nor cell type dependent, as ectopically expressing Tbx2 in a murine melanoma cell line also induce mitotic defects and polyploidy. These results have important implications for our understanding of the role of Tbx2 in tumorigenesis because polyploidy frequently precedes aneuploidy, which is associated with high malignancy and poor prognosis.

A prominent mechanism through which tumors develop resistance to DNA damaging chemotherapeutic agents is by cancer cells preferentially arresting in S-phase, thereby acquiring the ability to repair drug-induced DNA damage and consequently evading apoptosis. Combining DNA damaging therapies with targeted approaches that disrupt the DNA damage repair process may therefore prove to be more effective against such tumors. The developmentally important transcription factor TBX2 has been suggested as a novel anti-cancer drug target, as it is overexpressed in several cancers and possesses strong anti-senescence and pro-proliferative functions. Indeed, when TBX2 is silenced, we are able to reverse several features of transformation in both breast cancer and melanoma cells. We and others have previously showed that ectopic expression of TBX2 confers resistance to DNA damaging chemotherapeutic drugs, cisplatin and doxorubicin. However, whether endogenous TBX2 confers resistance to these DNA damaging agents in TBX2-driven cancers and the mechanism(s) by which this may occur have not been reported. To address this, we have silenced TBX2 in cisplatin-resistant breast cancer and melanoma cells and we show that knocking down TBX2 sensitizes the cells to cisplatin by disrupting the ATM-CHK2-p53 signalling cascade. Cell cycle analyses demonstrate that when TBX2 is knocked down there is an abrogation of an S-phase arrest but a robust G2/M arrest that correlates with a reduction in p-CHK2 and p53 levels. This prevents DNA repair resulting in TBX2 deficient cells entering mitosis with damaged DNA and consequently undergoing mitotic catastrophe. These results provide compelling evidence that targeting TBX2 in combination with chemotherapeutic drugs such as cisplatin could improve their efficacy and that it may be a viable treatment of TBX2-driven cancers. Citation Format: Serah Kimani, Sabina Wansbelen, Emily Davis, Jade Peres, Sharon Prince. The oncogenic TBX2 activates the ATM-CHK2-p53 axis to confer cisplatin resistance in breast cancer and melanoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B19.

The T-box transcription factors TBX2 and TBX3 are overexpressed in several cancers and are able to bypass senescence by repressing ARF and p21(WAF1/CIP1/SDII). Although these studies suggest that they may both contribute to the oncogenic process by repressing common targets, whether they have redundant or distinct roles in cancers where they are both overexpressed remains to be elucidated. Importantly, when Tbx2 function is inhibited in melanoma cells lacking Tbx3, the cells senesce, but whether this is possible in melanoma cells overexpressing both proteins is not known. An understanding of this issue may have important implications for the design of an effective pro-senescence therapy. In this study, the authors used a sh-RNA approach to knock down TBX2 and TBX3 individually in 2 human melanoma cell lines that overexpress both these factors and then examined their specific involvement in the oncogenic process. They demonstrate, using in vitro and in vivo cell proliferation, as well as colony- and tumor-forming ability and cell motility assays, that TBX2 and TBX3 have distinct roles in melanoma progression. In the tested lines, although TBX2 could promote proliferation and transformation and was required by primary melanoma cells for immortality, TBX3 was required for tumor formation and cell migration. These findings were reproducible in a human breast cancer cell line, which confirms that TBX2 and TBX3, although highly homologous, do not have redundant roles in the transformation process of cancers where they are both overexpressed. These results have important implications for the development of new cancer treatments and in particular for melanoma, which is a highly aggressive and intractable cancer.