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Background: All patients with pancreatic ductal adenocarcinoma (PDAC) experience disease progression after gemcitabine-nab-paclitaxel (GemPac) treatment with a median time to progression of just under 6 months. There is an urgent need for more effective combination therapies for this highly recalcitrant disease. Previously we showed that the selective inhibitor of nuclear export (SINE) selinexor (Sel) against XPO1 in combination with GemPac enhanced the survival of LSL-Kras G12D/+; Trp53 fl/+; Pdx1-Cre (KPC) mice. The Sel-GemPac combination also showed evaluable response in PDAC patients in a Phase I study. Here we identified key regulatory molecules involved in the synergy of the Sel-GemPac combination, utilizing KPC mouse tumors and patient biopsy samples. We also evaluated the efficacy of this combination in a Phase II study. Methods: The activity of Sel-GemPac was evaluated in KPC mouse derived 2D cell line and 3D spheroid models. RNA-seq was performed to evaluate transcriptional differences associated with synergy. The identified differentially expressed genes (DEG) were validated in vitro through siRNA mediated silencing, qPCR, cytotoxicity, and related assays. The molecular mechanism of synergy was also evaluated in KPC mice tumors and in biopsies from patients enrolled in the Phase I portion of the trial using spatial transcriptomic and proteomic analysis. The efficacy of Sel-Gem was evaluated in a Phase II study in patients with metastatic PDAC (NCT02178436). Results: In a KPC tumor derived cell line, Sel inhibited cell growth and suppressed spheroid formation. Sel synergized with GemPac (CI Conclusions: Collectively, our studies show that XPO1 may be a valid therapeutic target in PDAC and Sel-GemPac is a combination warranting further investigation. Citation Format: Md. Hafiz Uddin, Sahar F. Bannoura, Husain Y. Khan, Amro Aboukameel, Yiwei Li, Ibrahim Azar, Eliza W. Bael, Miguel Tobon, Donald Weaver, Steve Kim, Tanya Odisho, Amr Mohammed, Gregory Dyson, Rafic Beydoun, Ramzi M. Mohammad, Herbert Chen, Bassel El-Rayes, Philip A. Philip, Mohammad N. Al-Hallak, Asfar S. Azmi. Identification of key regulators for nuclear protein export inhibitor and chemotherapy combination in pancreatic cancer using spatial genomics approach [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 LB299.

Pancreatic cancer is a deadly disease that is increasing in incidence throughout the world. There are no clear causal factors associated with the incidence of pancreatic cancer; however, some correlation to smoking, diabetes and alcohol has been described. Recently, a few studies have linked the human microbiome (oral and gastrointestinal tract) to pancreatic cancer development. A perturbed microbiome has been shown to alter normal cells while promoting cancer-related processes such as increased cell signaling, immune system evasion and invasion. In this article, we will review in detail the alterations within the gut and oral microbiome that have been linked to pancreatic cancer and explore the ability of other microbiomes, such as the lung and skin microbiome, to contribute to disease development. Understanding ways to identify a perturbed microbiome can result in advancements in pancreatic cancer research and allow for prevention, earlier detection and alternative treatment strategies for patients.

Simple Summary Non-Hodgkin’s lymphomas (NHL) are cancers of the white blood cells. While some NHL subtypes, such as Diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), grow and spread aggressively, others, like follicular lymphoma (FL), are indolent in nature. Irrespective of how fast they grow, all NHL subtypes can spread to other organs in the body if not treated. In this study, we have demonstrated that the targeted inhibition of p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyl transferase (NAMPT) in different NHL subtypes by a novel, orally bioavailable, dual inhibitor KPT-9274 can lead to energy depletion, inhibition of cell proliferation, and ultimately apoptosis. KPT-9274 treatment shows potent anti-tumor effects in DLBCL and MCL subcutaneous xenograft models and enhances mice survival in a systemic FL model. Therefore, this study demonstrates the potential of targeting PAK4 and NAMPT by a small molecule inhibitor KPT-9274 for NHL therapy. Abstract Diffuse large B-cell lymphoma (DLBCL), grade 3b follicular lymphoma (FL), and mantle cell lymphoma (MCL) are aggressive non-Hodgkin’s lymphomas (NHL). Cure rates are suboptimal and novel treatment strategies are needed to improve outcomes. Here, we show that p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyl transferase (NAMPT) is critical for lymphoma subsistence. Dual targeting of PAK4-NAMPT by the Phase I small molecule KPT-9274 suppressed cell proliferation in DLBCL, FL, and MCL. Growth inhibition was concurrent with apoptosis induction alongside activation of pro-apoptotic proteins and reduced pro-survival markers. We observed NAD suppression, ATP reduction, and consequent cellular metabolic collapse in lymphoma cells due to KPT-9274 treatment. KPT-9274 in combination with standard-of-care chemotherapeutics led to superior inhibition of cell proliferation. In vivo, KPT-9274 could markedly suppress the growth of WSU-DLCL2 (DLBCL), Z-138, and JeKo-1 (MCL) sub-cutaneous xenografts, and a remarkable increase in host life span was shown, with a 50% cure of a systemic WSU-FSCCL (FL) model. Residual tumor analysis confirmed a reduction in total and phosphorylated PAK4 and activation of the pro-apoptotic cascade. This study, using various preclinical experimental models, demonstrates the therapeutic potential of targeting PAK4-NAMPT in DLBCL, FL, and MCL. The orally bioavailable, safe, and efficacious PAK4-NAMPT dual inhibitor KPT-9274 warrants further clinical investigation.

Simple Summary Pancreatic cancer is the fourth leading cause of cancer death in the United States and over 90% of the patients suffer from pancreatic ductal adenocarcinoma (PDAC). PDAC is the most lethal gastrointestinal malignancies and only 10% of the people survive more than 5 years, therefore, novel diagnostic, prognostic, and therapeutic strategies are an immediate necessity. Studies have demonstrated microRNAs in bodily fluids that are bound with membranes (exosomes) can act as stable biomarkers both for disease development and metastasis. The diagnostic, prognostic, as well as therapeutic roles of exosomal microRNAs in pancreatic cancer have been discussed in this review. Abstract Pancreatic cancer is the fourth leading cause of cancer death among men and women in the United States, and pancreatic ductal adenocarcinoma (PDAC) accounts for more than 90% of pancreatic cancer cases. PDAC is one of the most lethal gastrointestinal malignancies with an overall five-year survival rate of ~10%. Developing effective therapeutic strategies against pancreatic cancer is a great challenge. Novel diagnostic, prognostic, and therapeutic strategies are an immediate necessity to increase the survival of pancreatic cancer patients. So far, studies have demonstrated microRNAs (miRNAs) as sensitive biomarkers because of their significant correlation with disease development and metastasis. The miRNAs have been shown to be more stable inside membrane-bound vesicles in the extracellular environment called exosomes. Varieties of miRNAs are released into the body fluids via exosomes depending on the normal physiological or pathological conditions of the body. In this review, we discuss the recent findings on the diagnostic, prognostic, and therapeutic roles of exosomal miRNAs in pancreatic cancer.

Gastric cancer is a deadly disease with a low 5-year survival rate of 32%. This disease is characterized by high rates of chemo resistance and late initial detection. We have previously reported that inhibition of nuclear exporter protein Exportin 1 (XPO1/CRM1) with the small molecule inhibitor selinexor (KPT-330) is a viable therapeutic approach in gastric cancer. Inhibition of XPO1 blocks proliferation and suppresses tumor growth through nuclear retention of tumor suppressor proteins such as TP53. Besides nuclear retention, we also demonstrated that XPO1 inhibition leads to a perturbation of cancer specific microRNAs including microRNA-7974 downregulation (miR-7974) and microRNA-129-1-3p upregulation (miR-129-1-3p) within a small RNA sequencing study. In this study we evaluated the role of these microRNAs in gastric cancer perpetuation in respect to their naive functions in five distinct subtypes of gastric cancer. Our results show over-expression of miR-7974 and loss of miR-129-1-3p promotes gastric cancer growth and progression, based on specific subtype of disease. We have further found that the differential expression of these microRNAs leads to gastric cancer cellular changes including cell cycle alterations, upregulation of autophagy processes, chemoresistance and alterations in the cellular framework leading to more invasive phenotypes. Validation with normal gastric epithelial cells revealed these microRNAs are relevant as they are perturbed in gastric cancer as well as the proteins in which they modulate. Finally, we have validated treatment of gastric cancer cells with the XPO1 inhibitor selinexor altered the expression of miR-7974 and miR-129-1-3p differently, based on subtype, as we have previously observed in small RNA sequencing screen, leading to a reversal of the pro-cancerous phenotypes previously observed. In vivo investigation is currently ongoing to identify whether we can capture either miR-7974 or miR-129-1-3p within the serum as further evidence of microRNA perturbation as a predictive biomarker for selinexor efficacy. Citation Format: Rachel E. Sexton, Amro Aboukameel, Yiwei Li, Husain Y. Khan, Md Hafiz Uddin, Trishyan Kashyap, Yosef Landesman, Anteneh Tesfaye, Asfar S. Azmi. Utilizing small noncoding RNAs as biomarkers for selinexor treatment in gastric 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 5832.

Background: The majority of pancreatic ductal adenocarcinoma (PDAC) patients experience disease progression after Gemcitabine-nab-Paclitaxel (GemPac) treatment indicating the need for more effective combination therapies for this highly recalcitrant disease. Earlier we showed that nuclear exporter protein exportin 1 (XPO1) is a valid therapeutic target in PDAC and selective inhibitor of nuclear export (SINE) selinexor (Sel), synergistically enhances the efficacy of GemPac in pancreatic cancer cells, spheroids, patient derived tumors and showed evaluable response in PDAC patients in a Phase I study. Here we investigated the mechanisms of this synergy utilizing PDAC cellular models in vitro and LSL-Kras G12D/+; Trp53 fl/+; Pdx1-Cre (KPC) mouse model. Methods: We used single-nuclei RNA sequencing in KPC tumors, and poly A RNA sequencing in PDAC cells post sel-Gem-Pac treatment. Prioritized sn-seq and RNA-seq identified molecules were validated in in vitro or in the PDAC patient samples through siRNA mediated silencing, quantitative gene expression, cytotoxicity and related assays. Results: In KPC model, selinexor-GemPac caused statistically significant enhancement in survival compared to controls (p less than 0.05). Molecular analysis of residual KPC tumors showed re-organization of tumor stromal architecture, suppression of proliferation and nuclear retention of tumor suppressors. Histopathological analysis for Ki67 suggests comparatively lower rate of proliferation and enhanced nuclear accumulation of FOXO3a in the treated mice. Loosening of stroma and lowering of collagen fiber is also evident in treated mice from Picrosirius and Masson’s trichrome staining. Additionally, the KPC derived primary tumor cells showed reduced colony formation upon combination treatment. Sn-seq showed reduction in tumor cell heterogeneity (less cell clusters based on gene expression), CDK8, CD44 and Camk1d stem cell and EMT related genes alongside significant alterations in mitochondrial signaling after combination treatment. In cellular models, GO and GSEA analyses of RNA-seq data predicted DNA templated regulation of transcription and DNA replication associated genes in Sel or Sel-GemPac mediated growth inhibition. The top downregulated genes including drug resistant related gene PFN1 and HSP90 identified in Sel or Sel-GemPac treated PDAC cells from RNA-seq data were validated using RNAi and confirmed their role in synergy. Conclusions: Taken together Selinexor in combination with Gemcitabine-nab-Paclitaxel enhanced the survival of KPC mice through inhibition of well-connected tumor promoting molecules resulting in broad penetration in PDAC supporting signaling. A phase II study involving selinexor-gemcitabine combination in patients with metastatic PDAC is ongoing. Citation Format: Md. Hafiz Uddin, Sahar Bannoura, Yiwei Li, Husain Y. Khan, Amro Aboukameel, Mohammed N. Al-Hallak, Rafic Beydoun, Steve Kim, Yosef Landesman, Ramzi M. Mohammad, Anthony Shields, Asfar S. Azmi. Nuclear protein export inhibitor selinexor chemotherapy combination for pancreatic cancer therapy [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 5330.

Metastatic pancreatic neuroendocrine tumors (PNET) remain an unmet clinical problem. Chronologic treatment in PNETs includes observation (watchful protocol), surgery, targeted therapy, and chemotherapy. However, increasing evidence illustrates that the outcomes of targeted therapeutic options for the treatment of advanced PNETs show minimal response. The FDA-approved mTOR inhibitor everolimus does not shrink these tumors. It only delays disease progression in a subset of patients, while a significant fraction acquires resistance and shows disease progression. Thus, there is a need for more effective targeted approaches to sensitize PNETs to everolimus for better treatment outcomes. Previously, we showed that mTOR regulator p21 activated kinase 4 (PAK4) and nicotinamide adenine dinucleotide biosynthesis enzyme nicotinamide phosphoribosyl transferase (NAMPT) were aberrantly expressed in PNET tissue and promoted everolimus resistance. In this report, we demonstrate that PAK4-NAMPT dual inhibitor KPT-9274 can synergize with everolimus (growth inhibition, colony suppression, and glucose uptake assays). KPT-9274-everolimus disrupted spheroid formation in multiple PNET models. Molecular analysis showed alteration of mTORC2 through downregulation of RICTOR as a mechanism supporting synergy with everolimus in vitro. KPT-9274 suppressed β-catenin activity via inhibition of PAK4, highlighting the cross-talk between Rho GTPases and Wnt signaling in PNETs. KPT-9274, given at 150 mg/kg in combination with sub-MTD everolimus (2.5 mg/kg), significantly suppressed two PNET-derived xenografts. These studies bring forward a well-grounded strategy for advanced PNETs that fail to respond to single-agent everolimus.

Introduction: The discovery of compounds that can bind covalently to KRASG12C and lock it in its inactive GDP-bound conformation has opened a window of opportunity to selectively target KRASG12C. Such agents have shown promising results in preclinical tumor models and recently in clinical trials as well. However, factors such as feedback reactivation or bypass of KRAS dependence are known to limit antitumor activity of KRAS inhibitors. This necessitates the need for combination approaches that can potentially sensitize tumors to KRAS inhibitors when co-targeted. In this study, we have tested two potent KRASG12C inhibitors, MRTX1257 and AMG510, on PDAC and NSCLC cell lines both as single agents and also in combination with nuclear transport inhibitor, Selinexor (KPT-330/XPOVIO). Methods: Cell growth inhibition was determined by MTT assay. Drug synergy analysis was performed and the isobolograms were generated using CalcuSyn 2.1 software (Biosoft, Cambridge, UK). Spheroid disintegration assay, clonogenic assay, immunofluorescence assay, and Western blotting were performed by standard methods. Results: MRTX1257 and AMG510, when combined with KPT-330, synergistically inhibited (Combination Index values less than 1) the proliferation of KRASG12C mutant cancer cell lines, namely MiaPaCa-2 (PDAC) and NCI-H2122 (NSCLC). Such an effect was observed neither in KRASWT (NCI-H1650) nor KRASG12D mutant (Panc-1) cells, suggesting that the synergistic effect on cell viability was specific to KRASG12C. Combined treatment of KPT-330 with either MRTX1257 or AMG510 resulted in enhanced disintegration of MiaPaCa-2 and NCI-H2122 spheroids, indicating the efficacy of the drug combinations in a 3-D cell growth model. In addition, similar synergistic action was also seen in a clonogenic assay, where MRTX1257 and AMG510 reduced the number and size of colonies formed by MiaPaCa-2 cells and KPT-330 further augmented this effect. KPT-330 also potentiated the MRTX1257- or AMG510-induced inhibition of KRAS-dependent signaling targets such as ERK1/2 phosphorylation. Mechanistically, we observed that KPT-330 caused nuclear retention of ETS repressor factor (ERF), a transcriptional repressor that is exported upon phosphorylation by ERK. Given that XPO1 transports hundreds of proteins, we are currently evaluating the global changes in RAS pathway proteins using high throughput approaches. Such studies are anticipated to unveil the molecular underpinnings of the effectiveness of this co-targeting approach that would help advance this combination of nuclear transport inhibitor with KRASG12C inhibitors to be tested in the clinic. Conclusion: In conclusion, we have demonstrated that the inhibitor of nuclear transport protein XPO1 has the ability to potentiate the anticancer activity of KRASG12C inhibitors in in vitro preclinical models of PDAC and NSCLC. Citation Format: Husain Y. Khan, Md. Hafiz Uddin, Yue Zhang, Yosef Landesman, Ammar Sukari, Misako Nagasaka, Asfar S. Azmi. Inhibition of nuclear transport protein XPO1 potentiates the effect of KRASG12C inhibitors [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 1058.

Although pluripotent stem cell (PSC) therapy has advantages for clinical applications because of the self-renewal and multi-lineage differentiation abilities of PSCs, it also has disadvantages in terms of the potential for PSCs to undergo malignant transformation or unexpected differentiation. The prevention of teratoma formation is the largest hurdle of all. Despite intensive studies that have investigated ways to block teratomas, such methods have yet to be further developed for clinical use. Here, a new approach has focused on exerting anti-tumorigenic effects using a novel mica fine particle (MFP) designated STB-HO. Treatment with STB-HO regulated pluripotency- and apoptosis-related genes in differentiating human embryonic stem (hES) cells, while there is no effects in undifferentiated hES cells. In particular, STB-HO blocked the anti-apoptotic gene BIRC5 and activated p53, p21 and the pro-apoptotic proteins Bim, Puma and p-Bad during early spontaneous differentiation. Moreover, STB-HO-pretreated differentiating hES cells did not give rise to teratomas following in vivo stem cell transplantation. Our in vitro and in vivo results suggest a method for teratoma prevention in the context of PSC-derived cell transplantation. This novel MFP could break through the limitations of PSC therapy.

Mitochondrial biogenesis (MtBIO) is the utmost requisite for cell’s replication and survival, and it has been found to be involved in chemoresistance. The chemoresistance is the foremost obstacle in the treatment of patients with ovarian cancer. Several studies showed that modulation of MtBIO can lead to cancer cell death. Therefore targeting MtBIO in ovarian cancer could be a promising therapeutic approach possibly through overcoming the chemoresistance. Potential targets of MtBIO will be discussed focusing on ovarian cancer in this review.

Cisplatin (CDDP) resistance is the foremost obstacle in the treatment of ovarian cancer patients. According to the cancer stem cell hypothesis, the recurrence and chemoresistance are presumed to be linked to cancer stem/progenitor cells. Here, we investigated the cancer stem cell like phenotypes and mechanism of chemoresistance in CDDP resistant ovarian cancer cells. A well-established CDDP sensitive ovarian cancer cell line A2780 and its resistant clone A2780 Cp were used and we also developed a supra resistant clone (SKOV3 Cp) from a naturally CDDP resistant cell line SKOV3 by six shocks treatment. Sensitiveness to CDDP was evaluated by MTT assay and self renewal efficiency by sphere formation, noble agar and clonogenic assays. A few cancer hallmarks and stem-cell markers were characterized using flow cytometry. Molecular analysis was performed using quantitative PCR and western blotting. For the silencing of selected genes small interfering RNA (siRNA) based approaches was utilized. Both CDDP resistant A2780 Cp and supra resistant SKOV3 Cp cell lines showed significantly (P < 0.05) higher self-renewal ability in sphere formation, noble agar and clonogenic assays than their parental counterparts. They also showed significant resistance to apoptosis and sub-G1 arrest upon CDDP treatment. Stem cell marker ALDH1 positivity rates were higher both in A2780 Cp and SKOV3 Cp cell lines, quantified by ALDEFLUORTM assay kit using flow cytometry. Hoechst 33342 dye effluxing side populations were increased about five folds in A2780 Cp cells and two folds in SKOV3 Cp cells compared to A2780 and SKOV3 cells respectively. Among major stemness related genes (OCT4, SOX2, NANOG, NESTIN, BMI1, KLF4 and ALDH1A1) quantitative PCR analysis showed significantly higher expression of ALDH1A1 and KLF4 genes in both resistant and supra resistant cells than their parental clones. Of two genes, silencing ALDH1A1 in A2780 and A2780 Cp cells using siRNA greatly reduced the stem cell population as detected by ALDEFLUORTM assay and sensitized cells to CDDP. Moreover, silencing of ALDH1A1 reduce the level of its downstream target NEK-2. We also observed the downregulation of ABC transporters (ABCB1, ABCG2 and ABCC1) by silencing either ALDH1A1 or NEK-2 gene. Taken together, present study showed for the first time that stemness gene ALDH1A1 can be involved in CDDP resistance via the upregulation of NEK-2 in ovarian or solid cancer. Citation Format: Md. Hafiz Uddin, Yong Sang Song. Association of ALDH1A1 with cisplatin resistance via modulation of NEK-2 in ovarian cancer stem-like cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2511.