Fernández-Zapico, Martín E.

Abstract Using exome sequencing for biomarker discovery and precision medicine requires connecting nucleotide-level variation with functional changes in encoded proteins. However, for functionally annotating the thousands of cancer-associated missense mutations, or variants of uncertain significance (VUS), purifying variant proteins for biochemical and functional analysis is cost-prohibitive and inefficient. We describe parallel functional annotation (PFA) of large numbers of VUS using small cultures and crude extracts in 96-well plates. Using members of a histone methyltransferase family, we demonstrate high-throughput structural and functional annotation of cancer-associated mutations. By combining functional annotation of paralogs, we discovered two phylogenetic and clustering parameters that improve the accuracy of sequence-based functional predictions to over 90%. Our results demonstrate the value of PFA for defining oncogenic/tumor suppressor functions of histone methyltransferases as well as enhancing the accuracy of sequence-based algorithms in predicting the effects of cancer-associated mutations.

Abstract Cachexia is a multifactorial syndrome characterized by extensive body weight, lean muscle and fat mass loss. Cachexia worsens cancer patient prognoses and contributes to poor chemotherapy tolerance and response rates. About 80% of pancreatic cancer patients suffer from cachexia and one-third die due to cachexia-related complications such as respiratory failure and cardiac arrest. Although there has been considerable research into cachexia mechanisms and interventions, there are, to date, no FDA-approved cachexia therapies. A major contributing factor could be the failure of animal models to accurately recapitulate the human condition. In this study, we generated an aged murine model of pancreatic cancer cachexia to compare cachexia progression in young versus aged tumor-bearing mice. Comparative transcriptomic analyses of the muscles identified 3-methyladenine (3-MA), a known autophagy inhibitor, as a candidate anti-wasting compound. In vitro analyses confirmed anti-wasting effects while in vivo analysis demonstrated that 3-MA could rescue muscle wasting and suppress tumor growth. Surprisingly, transcriptome analyses of 3MA-treated tumor cells did not reveal autophagy-related signatures but instead implicated Perp as a novel 3-MA target. Probing further, we 1) observed significantly higher expression of Perp in pancreatic cancer cell lines compared to control cells, 2) noted a survival disadvantage associated with elevated PERP in the TCGA database, 3) found that PERP knockdown in cancer cells led to reduced tumor proliferation, and 4) confirmed that 3-MA-associated Perp reduction inhibited tumor growth. Moreover, we also demonstrate that survival benefits associated with 3MA is independent of tumor progression. Finally, we queried Perp expression in human tumor samples and observed increased Perp expression in adenocarcinoma regions compared to tumor adjacent tissue. Taken together, we report a novel mechanism linking 3-MA to Perp inhibition, and further implicate Perp as a novel tumor promoting factor in pancreatic cancer. Citation Format: Aneesha Dasgupta, Paige C. Arneson-Wissink, Rebecca E. Schmitt, Dong Seong Cho, Alexandra M. Ducharme, Tara L. Hogenson, Eugene W. Krueger, William R. Bamlet, Lizhi Zhang, Gina Razidlo, Martin E. Fernandez-Zapico, Jason D. Doles. Identification of perp as a tumor promoter in pancreatic cancer via the utilization of an aged model of cancer cachexia [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 3995.

Abstract Despite development of personalized treatments GI cancer like pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) remain dismal outcomes. While some patients are potentially curable with surgery and chemotherapy, most unresectable cases are invariably lethal. To advance patient care, we rely on clinical trials to test new therapeutic approaches. Here, we assume that the highest dose of a drug that does not cause severe toxicities is optimal for cancer treatment. However, this treatment strategy, in many cases, may be more harmful for the patient. Adopting principles from the field of evolutionary biology and pest management could offer more effective strategies that can be used for patients with metastatic PDAC and CRC. Resistance mechanisms are assumed to come with an energy cost, making the organism/cell less fit than others in the population. When high doses of treatment are applied, it results in a significant selection gradient by eliminating sensitive populations, freeing resources and facilitating resistant population growth. Such evolutionary dynamics and interactions within the tumor microenvironment can be modeled and exploited, in the form of adaptive strategies, for treatment of CRC and PDAC. It is our central hypothesis that integration of evolutionary dynamics and into PDAC and CRC treatment will delay emergence of resistance, enhance quality of life, and improve clinical outcomes. Our preliminary observations in co-cultured drug sensitive and resistant cell containing spheroids show that a higher proportion of drug sensitive cells inhibits the proliferation of drug resistant cells over time, in the presence of chemotherapeutic agents 5-FU and Oxaliplatin. We also demonstrate a role for the MLL family of histone methyltransferases in the epigenetic regulation of genes involved in chemoresistance. RNA-seq data has shown differential gene expression in metabolic genes regulated by histone methyltransferase KMT2A, KM2TB and KMT2D knockout (KO) cells including Gstt1, CYP26B1 and PYGL. In addition to this, KMT2A, KM2TB and KMT2D KOs show differential sensitivity to chemotherapeutic agents Gemcitabine, SN-38, 5-FU and Oxaliplatin. Further, analysis of KMT2A, KM2TB and KMT2D KO cells will provide mechanistic insight of the epigenetic regulation of metabolic patterns in drug resistant cells. This strategy would provide an approach for reducing exposure to toxic drugs, improving patient quality of life and renewing hope for prolonged control of metastatic PDAC and CRC. This proposal represents a bedside-to-bench-to-bedside project; identifying a shortcoming in how we currently treat PDAC and CRC patients, developing solutions in the lab and ultimately translating such strategies to benefit patients. Citation Format: Glancis Luzeena Raja Arul, Ezequiel Tolosa, Ryan M. Carr, Martin E. Fernandez-Zapico. Modeling evolutionary therapy for GI malignancies [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B002.

AbstractImmune checkpoint inhibitors (ICIs), as a novel class of anticancer therapy, can be more efficacious and less toxic than chemotherapy, but their clinical success is confined to certain tumor types. Elucidating their targets, mechanisms and scope of action, and potential synergism with chemotherapy and/or targeted therapies are critical to widen their clinical indications. Treatment response to an ICI targeting programmed death‐1 (anti‐PD‐1) is sought to be understood here by conducting a preplanned correlative analysis of a phase II clinical trial in patients with small bowel adenocarcinoma (SBA). The cytolytic capacity of circulating immune cells in cancer patients using a novel ex vivo cytotoxicity assay is evaluated, and the utility of circulating biomarkers is investigated to predict and monitor the treatment effect of anti‐PD‐1. Baseline expression of Bim and NKG7 and upregulation of CX3CR1 in circulating T cells are associated with the clinical benefit of anti‐PD‐1 in patients with SBA. Overall, these findings suggest that the frequency and cytolytic capacity of circulating, effector immune cells may differentiate clinical response to ICIs, providing a strong rationale to support immune monitoring using patient peripheral blood.

Approximately 80% of pancreatic cancer patients suffer from cachexia, and one-third die due to cachexia-related complications such as respiratory failure and cardiac arrest. Although there has been considerable research into cachexia mechanisms and interventions, there are, to date, no FDA-approved therapies. A major contributing factor for the lack of therapy options could be the failure of animal models to accurately recapitulate the human condition. In this study, we generated an aged model of pancreatic cancer cachexia to compare cachexia progression in young versus aged tumor-bearing mice. Comparative skeletal muscle transcriptome analyses identified 3-methyladenine (3-MA) as a candidate antiwasting compound. In vitro analyses confirmed antiwasting capacity, while in vivo analysis revealed potent antitumor effects. Transcriptome analyses of 3-MA–treated tumor cells implicated Perp as a 3-MA target gene. We subsequently (a) observed significantly higher expression of Perp in cancer cell lines compared with control cells, (b) noted a survival disadvantage associated with elevated Perp , and (c) found that 3-MA–associated Perp reduction inhibited tumor cell growth. Finally, we have provided in vivo evidence that survival benefits conferred by 3-MA administration are independent of its effect on tumor progression. Taken together, we report a mechanism linking 3-MA to Perp inhibition, and we further implicate Perp as a tumor-promoting factor in pancreatic cancer.

Changes in nuclear shape have been extensively associated with the dynamics and functionality of cancer cells. In most normal cells, nuclei have a regular ellipsoid shape and minimal variation in nuclear size; however, an irregular nuclear contour and abnormal nuclear size is often observed in cancer, including pancreatic cancer. Furthermore, alterations in nuclear morphology have become the ‘gold standard’ for tumor staging and grading. Beyond the utility of altered nuclear morphology as a diagnostic tool in cancer, the implications of altered nuclear structure for the biology and behavior of cancer cells are profound as changes in nuclear morphology could impact cellular responses to physical strain, adaptation during migration, chromatin organization, and gene expression. Here, we aim to highlight and discuss the factors that regulate nuclear dynamics and their implications for pancreatic cancer biology.

The Hh/GLI signaling pathway was originally discovered in Drosophila as a major regulator of segment patterning in development. This pathway consists of a series of ligands (Shh, Ihh, and Dhh), transmembrane receptors (Ptch1 and Ptch2), transcription factors (GLI1–3), and signaling regulators (SMO, HHIP, SUFU, PKA, CK1, GSK3β, etc.) that work in concert to repress (Ptch1, Ptch2, SUFU, PKA, CK1, GSK3β) or activate (Shh, Ihh, Dhh, SMO, GLI1–3) the signaling cascade. Not long after the initial discovery, dysregulation of the Hh/GLI signaling pathway was implicated in human disease. Activation of this signaling pathway is observed in many types of cancer, including basal cell carcinoma, medulloblastoma, colorectal, prostate, pancreatic, and many more. Most often, the activation of the Hh/GLI pathway in cancer occurs through a ligand-independent mechanism. However, in benign disease, this activation is mostly ligand-dependent. The upstream signaling component of the receptor complex, SMO, is bypassed, and the GLI family of transcription factors can be activated regardless of ligand binding. Additional mechanisms of pathway activation exist whereby the entirety of the downstream signaling pathway is bypassed, and PTCH1 promotes cell cycle progression and prevents caspase-mediated apoptosis. Throughout this review, we summarize each component of the signaling cascade, non-canonical modes of pathway activation, and the implications in human disease, including cancer.

Abstract Background Colorectal cancers (CRCs) are intrinsically drug resistant tumors with frequent low or absent tumor cell PD-L1 expression. Previously, we reported that PD-L1 depletion in CRC cells can confer chemoresistance. Given that JNK signaling has been implicated in cell survival and drug resistance in CRC cells, we examined the role of JNK and its regulation in chemoresistance in CRC cells with loss of PD-L1. Materials & MethodsHuman CRC cell lines (RKO, DLD1 and HCT15) were utilized and parental and PD-L1 knockout/knockdown cells were generated. Cells were treated with a JNK1-3 inhibitor (JNK-IN-8) alone or combined with a MEK1/2 inhibitor (cobimetinib), CPT-11 or oxaliplatin. Protein/protein interaction was analyzed by immunoprecipitation (IP); protein/RNA interaction was analyzed by RNA IP assay. RNA level and half life were measured by QRT-PCR. Apoptosis was measured by cleaved caspase-3 and quantified by Annexin V labeling using FACS. Colony formation assay was performed. ResultsPD-L1 depletion in CRC cells was shown to enhance JNK activity that was due to reduced mRNA stability of the upstream CYLD deubiquitinase. Since PD-L1 competes with the ribonuclease EXOSC10 for binding to CYLD mRNA, loss of PD-L1 increases the interaction of EXOSC10 and CYLD mRNA resulting in its ribonuclease-mediated degradation and activation of JNK signaling. JNK activation increased BIM phosphorylation at Thr116 that promotes its sequestration by MCL-1 and BCL-2. Notably, a selective and irreversible JNK1-3 inhibitor (JNK-IN-8) reduces BIMThr116 phosphorylation and can release BIM from its sequestration by MCL-1 and BCL-2 to promote apoptosis. Furthermore, use of JNK-IN-8, an MCL-1 antagonist (AZD5991), or their combination in tumor cells lacking PD-L1 is shown to promote apoptosis and reduce long-term clonogenic survival induced by multiple anti-cancer drugs. As confirmation, knockdown of PD-L1 can confer drug resistance in a human colon cancer-derived organoid model that can be reversed by JNK-IN-8. Conclusion Tumor cells with low or absent PD-L1 expression show increased JNK activity that promotes BIM sequestration by MCL-1/BCL-2 to confer multiple drug resistance that can be reversed by a JNK inhibitor. Citation Format: Frank A. Sinicrope, Lei Sun, Arpad Patai, Tara L. Hogenson, Martin E. Fernandez-Zapico, Bo Qin. PD-L1-mediated resistance to chemotherapy is overcome by an irreversible JNK inhibitor in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1085.