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Kirsten Rat Sarcoma (KRAS) is a master oncogene involved in cellular proliferation and survival and is the most commonly mutated oncogene in all cancers. Activating KRAS mutations are present in over 90% of pancreatic ductal adenocarcinoma (PDAC) cases and are implicated in tumor initiation and progression. Although KRAS is a critical oncogene, and therefore an important therapeutic target, its therapeutic inhibition has been very challenging, and only recently specific mutant KRAS inhibitors have been discovered. In this review, we discuss the activation of KRAS signaling and the role of mutant KRAS in PDAC development. KRAS has long been considered undruggable, and many drug discovery efforts which focused on indirect targeting have been unsuccessful. We discuss the various efforts for therapeutic targeting of KRAS. Further, we explore the reasons behind these obstacles, novel successful approaches to target mutant KRAS including G12C mutation as well as the mechanisms of resistance.

Simple Summary Pancreatic cancer is the seventh leading cause of cancer related death worldwide. In the United States, pancreatic cancer remains the fourth leading cause of cancer related death. The lack of early diagnosis and effective therapy contributes to the high mortality of pancreatic cancer. Therefore, there is an urgent need to find novel and effective biomarkers for the diagnosis and treatment of pancreatic cancer. Long noncoding RNA, circular RNAs and piwi-interacting RNA are non-coding RNAs and could become new biomarkers for the diagnosis, prognosis, and treatment of pancreatic cancer. We summarize the new findings on the roles of these non-coding RNAs in pancreatic cancer diagnosis, prognosis and targeted therapy. Abstract Pancreatic cancer is an aggressive malignance with high mortality. The lack of early diagnosis and effective therapy contributes to the high mortality of this deadly disease. For a long time being, the alterations in coding RNAs have been considered as major targets for diagnosis and treatment of pancreatic cancer. However, with the advances in high-throughput next generation of sequencing more alterations in non-coding RNAs (ncRNAs) have been discovered in different cancers. Further mechanistic studies have demonstrated that ncRNAs such as long noncoding RNAs (lncRNA), circular RNAs (circRNA) and piwi-interacting RNA (piRNA) play vital roles in the regulation of tumorigenesis, tumor progression and prognosis. In recent years, increasing studies have focused on the roles of ncRNAs in the development and progression of pancreatic cancer. Novel findings have demonstrated that lncRNA, circRNA, and piRNA are critically involved in the regulation of gene expression and cellular signal transduction in pancreatic cancer. In this review, we summarize the current knowledge of roles of lncRNA, circRNA, and piRNA in the diagnosis and prognosis of pancreatic cancer, and molecular mechanisms underlying the regulation of these ncRNAs and related signaling in pancreatic cancer therapy. The information provided here will help to find new strategies for better treatment of pancreatic cancer.

Aberrant nuclear protein transport, often observed in cancer, causes mislocalization dependent inactivation of critical cellular proteins. Earlier we showed that over-expression of the nuclear export protein exportin 1 (XPO1) is linked to higher grade and Gleason score in metastatic castration resistant prostate cancer (mCRPC). We also showed that the selective inhibitor of nuclear export (SINE) compound selinexor and the second generation SINE eltanexor (KPT-8602) could suppress mCRPC growth, reduce androgen receptor (AR), and re-sensitize to androgen deprivation therapy. Here we evaluated the combination of KPT-8602 with three different PARP inhibitors (PARPi) namely olaparib, veliparib and rucaparib in a mCRPC model. Growth inhibition was determined by MTT assay. Drug synergy analysis was done and the isobolograms were generated from Calcusyn 2.1 software (Biosoft, Cambridge, UK). Other techniques include: colony formation assay, apoptosis determination by flow cytometry (annexin V-propidium iodide), real time RT-qPCR (SYBR green I), western blotting, immunofluorescence etc. For the determination of band density, NIH ImageJ 1.5Oi software was utilized. KPT-8602 synergized with all three studied PARPi (CI Taken together, this study revealed the therapeutic potential of a novel combination including second generation SINE compound KPT-8602 and PARP inhibitors for the growth inhibition of mCRPC cells. Pre-clinical xenograft studies testing the efficacy of SINE-PARPi as well as a phase II clinical study combining KPT-8602-PARPi is planned. Citation Format: Md. Hafiz Uddin, Yiwei Li, Amro Aboukameel, Husain Y. Khan, Vijendra Singh, Shriya Reddy, Suresh Kumar Balasubramanian, Yosef Landesman, Trinayan Kashyap, Elisabeth I. Heath, Asfar S. Azmi. Nuclear export inhibitor KPT-8602 synergize with PARP inhibitors in castration resistant metastatic prostate cancer [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 1091.

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.

It has been almost two decades since the first link between microRNAs and cancer was established. In the ensuing years, this abundant class of short noncoding regulatory RNAs has been studied in virtually all cancer types. This tremendously large body of research has generated innovative technological advances for detection of microRNAs in tissue and bodily fluids, identified the diagnostic, prognostic, and/or predictive value of individual microRNAs or microRNA signatures as potential biomarkers for patient management, shed light on regulatory mechanisms of RNA–RNA interactions that modulate gene expression, uncovered cell‐autonomous and cell‐to‐cell communication roles of specific microRNAs, and developed a battery of viral and nonviral delivery approaches for therapeutic intervention. Despite these intense and prolific research efforts in preclinical and clinical settings, there are a limited number of microRNA‐based applications that have been incorporated into clinical practice. We review recent literature and ongoing clinical trials that highlight most promising approaches and standing challenges to translate these findings into viable microRNA‐based clinical tools for cancer medicine. This article is categorized under:RNA in Disease and Development > RNA in Disease, microRNAs are short noncoding regulatory RNAs with important roles in cancer biology. microRNAs are informative biomarkers for early cancer detection and treatment decisions. microRNAs are being evaluated in clinical trials as new drugs for personalized cancer medicine.

Colorectal cancer (CRC) is a leading cause of cancer related deaths worldwide and the third leading cause of cancer related deaths in the United States with a 5-year survival rate of 63%. Depending on the stage of disease, there are differing rates of overall survival: Stage I (90%) vs. Stage IV (14%). There is a clear discrepancy between early and late stage disease due to a variety of factors including acquired genetic and tumor mutations and lack of initial symptoms leading to late stage detection. It is clear that further understanding into the mechanisms of CRC need to be explored in depth to improve overall survival rates. ATP binding cassette subfamily B member 5 (ABCB5) has been found to be pathogenic in a variety of diseases including melanoma and colorectal cancer but there has been few studies looking at the cellular consequences of ABCB5 upregulation in CRC cells. Using publicly accessible databases, we explored ABCB5 positivity in CRC cells and found novel interactions with 64 differentially expressed genes involved in cellular processes like modulation of inflammation, (XCL1, TNFSF4), enhanced cell signaling via interaction with serine protease inhibitors (SERPINB4, WFDC6) and downregulation of anti-apoptotic proteins (SPIN2A). Although ABCB5 upregulation is not currently targetable with pre-clinical compounds, our analysis revealed the genetic effects induced by ABCB5 upregulation are targetable, including evidence of sensitivity to the XPO1 inhibitor XPOVIO (selinexor). It is clear further pre-clinical investigation is needed to validate these findings.

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.

Liquid biopsy is now considered a valuable diagnostic tool for advanced metastatic non-small cell lung cancer (NSCLC). In NSCLC, circulating tumor DNA (ctDNA) analysis has been shown to increase the chances of identifying the presence of targetable mutations and has been adopted by many clinicians owing to its low risk. Serial monitoring of ctDNA may also help assess the treatment response or for monitoring relapse. As the presence of detectable plasma ctDNA post-surgery likely indicates residual tumor burden, studies have been performed to quantify plasma ctDNA to assess minimal residual disease (MRD) in early-stage resected NSCLC. Most data on utilizing liquid biopsy for monitoring MRD in early-stage NSCLC are from small-scale studies using ctDNA. Here, we review the recent research on liquid biopsy in NSCLC, not limited to ctDNA, and focus on novel methods such as micro RNAs (miRNA) and long non-coding (lncRNA).

Gastric cancer remains a major unmet clinical problem with over 1 million new cases in 2018 worldwide. It is the fourth most commonly occurring cancer in men and the seventh most commonly occurring cancer in women. A major fraction of gastric cancer has been linked to variety of pathogenic infections including but not limited to Helicobacter pylori (H. pylori) or Epstein Barr virus (EBV). Strategies are being pursued to prevent gastric cancer development such as H. pylori eradication, which has helped to prevent significant proportion of gastric cancer. Today, treatments have helped to manage this disease and the 5-year survival for stage IA and IB tumors treated with surgery are between 60% and 80%. However, patients with stage III tumors undergoing surgery have a dismal 5-year survival rate between 18% and 50% depending on the dataset. These figures indicate the need for more effective molecularly driven treatment strategies. This review discusses the molecular profile of gastric tumors, the success and challenges with available therapeutic targets along with newer biomarkers and emerging targets.

Highlights • Nucleo-cytoplasmic transport is crucial for the proper functioning of cells. • Studies targeting nucleo-cytoplasmic transport have chiefly focused on cancer. • XPO1 mediated protein or RNA export is necessary for viral replication. • XPO1 contributes to the immunopathology by activating inflammatory signaling. • Inhibition of nuclear export using SINE may have broad-spectrum antiviral effect., Coronavirus 2019 (COVID-19; caused by Severe Acute Respiratory Syndrome Coronavirus 2; SARS-CoV-2) is a currently global health problem. Previous studies showed that blocking nucleocytoplasmic transport with exportin 1 (XPO1) inhibitors originally developed as anticancer drugs can quarantine key viral accessory proteins and genomic materials in the nucleus of host cell and reduce virus replication and immunopathogenicity. These observations support the concept of the inhibition of nuclear export as an effective strategy against an array of viruses, including influenza A, B, and SARS-CoV. Clinical studies using the XPO1 inhibitor selinexor as a therapy for COVID-19 infection are in progress.

Pancreatic ductal adenocarcinoma (PDAC) remains a challenging disease that is mostly diagnosed late in the course of the illness. Unlike other cancers in which measurable successes have been achieved with traditional chemotherapy, targeted therapy, and, recently, immunotherapy, PDAC has proved to be poorly responsive to these treatments, with only marginal to modest incremental benefits using conventional cytotoxic therapy. There is, therefore, a great unmet need to develop better therapies based on improved understanding of biology and identification of predictive and prognostic biomarkers that would guide therapy. miRNAs are small noncoding RNAs that regulate the expression of some key genes by targeting their 3'-untranslated mRNA region. Aberrant expression of miRNAs has been linked to the development of various malignancies, including PDAC. A series of miRNAs have been identified as potential tools for early diagnosis, prediction of treatment response, and prognosis of patients with PDAC. In this review, we present a summary of the miRNAs that have been studied in PDAC in the context of disease biology.

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.

Background: KRASG12C inhibitors sotorasib and adagrasib have shown impressive results in clinical studies. These inhibitors efficiently block KRAS downstream effectors such as RAF, MEK, and ERK/p-ERK. However, factors such as feedback reactivation or bypass of KRAS dependence are emerging, resulting in the need for rational combination approaches. KRASG12C inhibitors have limited impact on the parallel oncogenic signaling driven by PI3K/AKT, its regulator molecules such as the Rho GTPases, and their effectors. This signaling pathway can collectively promote p-ERK re-activation thereby blunting the efficacy of KRASG12C targeted drugs. The p21-activated kinase (PAK) family are crucial effectors of the Rho family of GTPases and act as regulatory switches that control important cellular processes including motility, proliferation, and survival. PAK4 is the main effector of Rho GTPase, cell division control protein 42 homolog (Cdc42), and has an established role in mutant RAS-dependent tumor metastasis. In this study, we rationally targeted two parallel signaling pathways by combining KRASG12C inhibitors with the PAK4 inhibitor KPT-9274 in KRASG12C mutant PDAC and NSCLC in vitro and in vivo models. Methods: We evaluated the cytotoxicity and molecular profile of KRASG12C inhibitors in combination with PAK4 inhibitor KPT-9274 as well as positive control PF-3758309 in KRASG12C mutant 2D and 3D cellular/spheroid models. The anti-tumor activity of the combinations was evaluated in KRASG12C mutant cell line-derived xenograft. Tumor volumes were assessed throughout the treatment period using an ultrasound scanner. Results: PAK4 inhibitor KPT-9274 synergized with both sotorasib and adagrasib yielding suppressed growth of KRASG12C mutant PDAC and NSCLC cells in 2D and 3D cultures, but not in KRASwt, KRASG12V, and KRASG12D cell lines (NCI RASless MEFs). In addition, the combination reduced the clonogenic potential of KRASG12C mutant cancer cells. PAK4-KRASG12C inhibitor combinations resulted in enhanced and prolonged inhibition of KRAS downstream effector pathways. In xenograft studies, oral administration of KPT-9274 at sub-optimal dose (100 mg/kg QD x 5 x 3 weeks) and sotorasib at one-fourth of MTD (25 mg/kg QD x 5 x 3 weeks) demonstrated remarkable inhibition of KRASG12C mutant MiaPaCa-2 tumor burden (p Conclusion: This is the first study showing that KRASG12C inhibitors can synergize with PAK4 inhibitors. This combination can simultaneously target direct KRAS downstream effectors and the parallel PI3K/AKT oncogenic signaling warranting further clinical investigations. Citation Format: Husain Yar Khan, Sahar Bannoura, Hirva Mamdani, Amro Aboukameel, Yousef Mzannar, Yiwei Li, Mohammad Najeeb Al-Hallak, Ibrahim Azar, Steve Kim, Rafic Beydoun, Ramzi Mohammad, Yosef Landesman, Yue Zhang, Erkan Baloglu, William Senapedis, Ammar Sukari, Misako Nagasaka, Anthony Shields, Asfar S. Azmi. Anti-tumor activity of KRASG12C inhibitors is enhanced when combined with Cdc42 effector p21-activated kinase 4 targeting agents [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 5315.

e18688 Background:177Lu-Dotatate is FDA approved for advanced well differentiated (WD) midgut neuroendocrine tumors (NETs). However, questions remain about tolerability and efficacy in patients (pts) with higher grade disease, more than one prior line of treatment, and in non-ileal primaries. Methods: This is a retrospective analysis of pts with histologically proven WD metastatic GEP-NETs who received 177Lu-Dotatate at a single racially diverse urban cancer center. Patients had disease that progressed on systemic therapy that included somatostatin analog (SSA), and radiotracer uptake at disease sites using 68Gallium-Dotate PET/CT. ECOG performance score (PS) was 0 to 2. 177Lu-Dotatate was administered in 200 mCi doses every 8 weeks for a total of 4 cycles and a maximum of 800 mCi. Patients were followed with serial CT scan and/or abdominal MRI with best objective response per RECIST criteria. Results: Fifty-one pts were treated between December 2017 and August 2021. Thirty-eight pts completed a minimum 3-month follow-up imaging. Median age was 64 years, 59% were female, 66% identified as white, 26% as Black, 8% as other. Most frequent primary sites were small intestine (64%) and. All had metastatic disease involving the liver except for one pt. Median time from diagnosis to 177Lu-Dotatate was 51 months (3-201). CTCAE grade 3/4 hematologic toxicity was seen in 6 of 51 pts (12%). Grade 3/4 thrombocytopenia occurred in 3 pts, grade 3/4 leukopenia occurred in 2 pts and grade 3 anemia occurred in 1 pt. A single pt developed myelodysplastic syndrome 1 year after 177Lu-Dotatate. Non-white pts had significantly higher risk of grade 1 elevated creatinine at 38% vs 6% in those who identified as white (p = 0.015). Of the 37 pts who reached 3-month follow-up, 27% had partial response, 43% had a mixed/stable response, and 30% had progressive disease. Only gender demonstrated significant difference; with male gender associated with worse response categories (p = 0.001). Median OS was 3.09 years. Metastatic disease in the liver only was associated with significantly worse OS (p = 0.015) compared to liver and other tissues. The median PFS was 1.36 years. Metastatic disease in the liver only was associated with significantly worse PFS (p = 0.043). Higher tumor grade neared significant association with worse PFS (p = 0.053). There was no difference in PFS by race, primary tumor site (pancreatic vs small bowel), PS, prior treatment history or time to first 177Lu-Dotatate. Conclusions: Diverse patients with WD GEP-NETs treated at this single urban cancer center with 177Lu-Dotatate demonstrated acceptable toxicity. Median PFS and OS were inferior to reported outcomes in NETTER-1 probably because of differences in treatment timing and patient populations. Possible variables associated with difference in outcome include pattern of metastatic disease and tumor grade.

e21149 Background: P21-activated kinase 4 (PAK4) is a crucial effector of the Rho GTPases. It acts as a regulatory switch that controls a wide range of cellular functions and plays a pivotal role in cancer progression and metastasis. Very little is known about the expression and prognostic value of PAK4 in NSCLC. Methods: 17,689 NSCLC tumor samples were analyzed using next-generation sequencing (NextSeq, 592 Genes, or WES NovaSeq), immunohistochemistry (IHC), and whole transcriptome sequencing (NovaSeq) (Caris Life Sciences, Phoenix, AZ). PD-L1 expression was analyzed by IHC (Dako 22c3; PD-L1 positive: TPS >1%). TMB was measured by counting all somatic mutations found per tumor (TMB-high: >10 mutations/MB). Tumors with PAK4-high (H) and PAK4-low (L) expression were classified as those in top quartile and bottom 3 quartiles, respectively. Immune cell infiltrates were calculated by Quantiseq. Significance was determined using chi-square and Wilcoxon rank sum test and adjusted for multiple comparisons (q-value < 0.05). Survival was extracted from insurance claims data and calculated from the time of tissue collection to the last contact using Kaplan-Meier estimates. Results: There was no difference in median age, gender, smoking status, and histologic distribution between PAK-H and PAK-L tumors. Compared to PAK4-L, the PAK4-H was associated with higher frequency of co-mutations in TP53 (76.3% vs 63.9%, p < 0.0001) and RB1 (13.6% vs 8.1%, p < 0.0001). PAK4-H tumors were associated with greater genomic loss of heterozygosity (24.1% vs 16.4%, p < 0.0001), and expression of immune checkpoint genes ( LAG3, PDCD1, PDCD1LG2, CD274, IDO1, CTLA4, CD80, HAVCR2; p < 0.05). KRAS (including KRAS G12C) , BRAF, STK11, and EGFR mutations, and ALK and ROS1 fusions were less frequent in PAK4-H tumors (p < 0.001). A greater proportion of PAK4-H tumors were TMB-H (40.3% vs 33.3%, p < 0.0001) and PD-L1 negative (48.2% vs 40.8%, p < 0.001). PAK4-H tumors had lower infiltration of B cells, M1/M2 macrophages, CD8+ T-cells, and Tregs (p < 0.001). Overall survival (OS) was inferior among patients with PAK4-H tumors (median, 14.9 vs 21.5 months, HR = 1.28, 95% CI, 1.21-1.36, p < 0.001), which was driven by adenocarcinoma histology. Survival with immunotherapy was also inferior in patients with PAK4-H adenocarcinoma (median, 23.6 vs 30.3 months, HR = 1.23, 95% CI, 1.02-1.48, p = 0.03), but not squamous cell carcinoma. A similar trend in survival was noted in patients who received EGFR- and ALK inhibitors. Conclusions: PAK4-H expression in NSCLC is associated with a higher frequency of alterations predisposing to genomic instability, differentially modulated immune phenotype, and a lower frequency of actionable genomic alterations. Patients with PAK4-H adenocarcinoma have inferior OS, and survival with immunotherapy and targeted therapy. PAK4-H expression defines a subgroup of patients with un unmet need for novel treatment strategies.

Despite many efforts to improve the outcome of pancreatic ductal adenocarcinoma (PDAC), its prognosis remains poor, which is mostly related to late diagnosis and drug resistance. Improving systemic therapy is considered the major challenge in improving the outcome of this disease.This review covers novel chemotherapy and targeted agents in the treatment of PDAC, with a focus on advanced stage disease.Current frontline therapies used in the treatment of patients with PDAC with favorable performance status are gemcitabine (GEM) and nab-paclitaxel or 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX). PDAC has a number of genetic mutations that may explain its biological behavior, such as KRAS, p53 and CDK2NA, which occur in more than 90% of cases. Unfortunately, to this day, a specific targeting agent to any of those frequent gene mutations is lacking. Emerging areas of targeted therapies include the DNA repair, stroma, metabolism, and stem cells. Immunotherapy with either vaccines or immune checkpoint inhibitors has not produced any significant improvements in outcome of PDAC. Incorporating different approaches in therapy, including conventional, immunological, and others, is key in offering patients with the best possible care.

Emerging studies have shown that the expression of AR splice variants (ARv) lacking ligand-binding domain is associated with castrate-resistant prostate cancer (CRPC) and higher risk of tumor metastasis and recurrence. Nuclear export protein XPO1 regulates the nuclear localization of many proteins including tumor suppressor proteins. Increased XPO1 in prostate cancer is associated with a high Gleason score and bone metastasis. In this study, we found that high expression of AR splice variant 7 (AR-v7) was correlated with increased XPO1 expression. Silencing of XPO1 by RNAi or treatment with Selective Inhibitor of Nuclear Export (SINE) compounds selinexor and eltanexor (KPT-8602) down-regulated the expression of AR, AR-v7 and ARv567es at mRNA and protein levels. XPO1 silencing also inhibited the expression of AR and ARv regulators including FOXA1, Src, Vav3, MED1 and Sam68, leading to the suppression of ARv and AR target genes, UBE2C and PSA. By targeting XPO1/ARv signaling, SINE suppressed prostate cancer (PCa) growth in vitro and in vivo and potentiated the anti-cancer activity of anti-AR agents, enzalutamide and abiraterone. Therefore, XPO1 inhibition could be a novel promising agent used in combination with conventional chemotherapeutics and AR-targeted therapy for the better treatment of PCa, especially CRPC. ispartof: Oncotarget vol:9 issue:82 pages:35327-35342 ispartof: location:United States status: Published online

Paclitaxel (PTX) encapsulated in albumin (Abraxane®) is an FDA approved frontline nano-formulation for treating advance metastatic pancreatic, lung and breast cancers. Currently in clinic, Abraxane® is being used as a one of the components of combination therapy regimens. On the other hand, difluorinated curcumin (CDF) is a novel and potent synthetic curcumin analogue that is being evaluated for several malignancies including pancreatic, liver, ovarian and breast cancers. To improve the bioavailability and targeting ability of hydrophobic PTX and CDF, we have encapsulated them in folic acid decorated bovine serum albumin nanoparticles, namely FA-BSA-PTX and FA-BSA-CDF, respectively. Both the formulations yielded uniform nano-sized particles with smooth surface morphology, negative surface potential and high drug loading efficiency. Due to heterogeneity and complexity of several cancers, combination regimens are becoming standard arsenals against several deadly cancers. To evaluate the synergistic anti-cancer effect of PTX and CDF, we assessed the combination therapy using intravenously administrable folate decorated albumin bio-conjugate nanoparticles against folate overexpressing ovarian and cervical cancers. Our results demonstrate that combination of FA-BSA-CDF with FA-BSA-PTX produced synergistic anticancer effect, augmented due to folate receptor mediated targeted uptake as well as induction of apoptosis. In conclusion, our preliminary studies show a promising nanomedicine platform for combination therapy for leading gynecological tumor, such as ovarian and cervical cancer.

Novel treatment strategies for pancreatic ductal adenocarcinoma (PDAC) are desperately needed. Local tumor progression is a cause of significant morbidity and mortality in patients with surgically unresectable disease. Often, regional anatomic structures limit the total doses of radiation therapy (RT) that can be safely delivered. Conventional doses of concurrent chemotherapy and RT (chemo-RT) have shown suboptimal results in local control of disease, progression free survival, and overall survival. Therefore, novel and effective approaches to enhance the efficacy of RT are urgently needed to improve overall survival in unresectable PDAC. Metabolic reprogramming enables cancer cells to adjust their metabolism to support increased energy requirements associated with continuous growth and proliferation. Indeed, metabolic reprogramming is a hallmark of PDAC and is associated with increased tumor cell plasticity and chemo-RT resistance. Cancer-cell mitochondria are key regulators of deranged tumor metabolism and have been shown to guide molecular pathways involved in radio-resistance. There is also expanding data that the presence of metabolites modulates the response of cancer cells to RT primarily by impacting the ability to repair DNA. This makes them an optimal candidate for novel radiosensitization strategies, as these characteristics are unique to PDAC cells, and are limited in normal cells. CPI-613, is an analog of lipoic acid which inhibits pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (α-KGDH), thereby disrupting mitochondrial metabolism leading to selective tumor cell killing. The drug has demonstrated significant clinical activity in patients with metastatic PDAC in combination with standard of care chemotherapies. It remains unknown as to the efficacy in patients treated with concurrent chemo-RT. Here we show that combined treatment of RT (2 and 10 Gy) with CPI-613 (used at 200 and 300 μM) causes superior inhibition of pancreatic cancer cell growth (MTT assay and colony formation assay). In addition, we demonstrate enhanced apoptosis (Annexin V FITC and 7AAD assay) of PDAC cells when treated with a combination of RT and CPI-613. Molecular analysis revealed superior inhibition of PDH and α-KGDH at the protein level. Targeted metabolomic analysis on PDAC cells post CPI-613-RT treatment revealed alterations in key mitochondrial metabolites, leading to these findings. These results indicate broader target engagement by this combination treatment, indicating the sensitization of CPI-613 treated PDAC cells to radiotherapy at doses as low as 2 Gy. Furthermore, in our preclinical cellular models, a combination treatment of CPI-613 with either Gemcitabine or 5-FU has shown synergistic effects on the proliferation of PDAC cells. Pre-clinical anti-tumor efficacy of the CPI-613-RT and CPI-613-RT-chemo using subcutaneous and orthotopic PDAC models is planned. Our results bring forward a novel combination of CPI-613-RT that warrants further pre-clinical and early phase clinical investigations. Citation Format: William A. Hall, Husain Y. Khan, Mandana Kamgar, Susan Tsai, Kathleen Christians, Douglas B. Evans, Philip Philip, Callisia Clarke, Ben George, Beth Erickson, Asfar S. Azmi. Targeting cellular metabolism with CPI-613 sensitizes pancreatic cancer cells to radiotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-024.

Aggressive non-Hodgkin's lymphomas (NHLs) [diffuse large b-cell lymphoma (DLBCL), grade 3b follicular lymphoma, and mantle cell lymphoma (MCL)] have dismal cure rates, especially in the relapsed/refractory setting, substantiating the need for novel treatment strategies to improve outcomes. Here we show that p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyltransferase (NAMPT) are essential for lymphoma subsistence and dual-targeting PAK4-NAMPT by the small molecule inhibitor KPT-9274 (orally bioavailable phase 1 compound), previously shown to be cancer cell-selective, acts by energy depletion, inhibiting cell proliferation and eventual apoptosis as a reasonable treatment strategy for aggressive NHLs. To demonstrate PAK4-NAMPT is essential for lymphoma survival, we showed increased PAK4 expression in primary NHL tissue (n=94) than in normal lymph nodes (n=8) (Fig1A). Lymphoma cell lines were sensitive to the PAK4/NAMPT dual inhibitor KPT-9274 (induced apoptosis with an EC 50 of 95.17 nM in WSU-DLCL2 and 13.9 nM in WSU-FSCCL cells). Similar results were obtained with positive controls PAK4 [PF-3578309] and NAMPT specific inhibitors [FK-866]. RT-PCR confirmed PAK4 and NAMPT down-expression in KPT-9274 treated WSU-FSCCL cells. Western blot of WSU-DLCL2 and WSU-FSCCL cells treated with KPT-9274 showed significant reductions in BCL-2 and enhanced cleaved-Caspase-3 and cleaved-PARP expression. We then assessed the impact of KPT-9274 on the cellular ATP and NAD pool since NAMPT inhibition can suppress both and induce cell death. NAD/NADH and ATP levels decreased in both cell lines [WSU-DLCL2 & WSU-FSCCL], with increasing KPT-9274 concentrations (Fig.1B&1C). We then showed KPT-9274 to synergize with the standard of care chemotherapy cyclophosphamide, vincristine, and adriamycin used for NHL management (decreased IC25 and IC50 in several combination treatments). In lymphoma xenografts, mice with transplanted WSU-DLCL2 fragments showed ~50% reduction in tumor volume (p 150-day increase in host life span compared to control and 3/6 mice were cured) (Fig.1F). After day 150, the remaining mice were dissected to confirm cures. Blood smears drawn from mice post three weeks of KPT-9274 treatment showed a significant reduction of circulating WSU-FSCCL cells validating its efficacy systemically (Fig.1G). Since exogenous niacin can regenerate NAD regulated by NAMPT in a rate-limiting step, the impact of niacin co-dosing with KPT-9274 was evaluated on tumor growth using the Z-138 MCL tumor xenograft model in nude mice. Tumor-bearing mice were treated with vehicle, KPT-9274 (200 mg/kg BID), niacin (30 mg/kg), or a combination of KPT-9274/niacin. All groups that received KPT-9274 treatment showed reductions in tumor size, while the vehicle control and niacin alone groups showed a rapid increase in tumor volume. Both KPT-9274 and combo groups showed a significant decrease in tumor growth compared to control (p=0.0002); however, there were no statistically significant differences between the KPT-9274 and the KPT-9274/niacin combo (Fig.1H). Similarly, in another MCL xenograft model, KPT-9274 resulted in a statistically significant reduction in JeKo-1 tumors compared to control (p = 0.0059). Niacin did not affect the tumor growth suppression induced by KPT-9274 treatment. No animals died in this study. Thus, supplementing niacin with KPT-9274 may alleviate the adverse effect without compromising the efficacy of the treatment. Also, KPT-9274 treated Z-138 residual tumors showed a reduction in PAK4, GEF-H1, paxillin, vinculin, cyclin D1, Dvl2, and Ki67 and enhancement in apoptosis by IHC staining (Fig.1I). The anti-tumor potential of KPT-9274 ± niacin in several aggressive lymphoma models strongly supports its efficacy in this setting. It strengthens our phase I study design to evaluate safety and tolerability and anti-tumor activity in patients with advanced solid malignancies or NHL (NCT02702492). Figure 1 Figure 1. Disclosures Balasubramanian: Servier Pharmaceuticals: Research Funding. Zonder: Caelum Biosciences: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Amgen: Consultancy; Alnylam: Consultancy; Regeneron: Consultancy; Janssen: Consultancy; Intellia: Consultancy. Senapedis: Karyopharm Therapeutics: Ended employment in the past 24 months, Patents & Royalties; Omega Therapeutics: Current Employment; Restorbio: Other: Personal fees. Baloglu: Karyopharm Therapeutics: Current Employment, Other: Personal fees. Shah: Karyopharm Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Landesman: Karyopharm Therapeutics: Current Employment, Current equity holder in publicly-traded company. Shacham: Karyopharm: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: (8999996, 9079865, 9714226, PCT/US12/048319, and I574957) on hydrazide containing nuclear transport modulators and uses, and pending patents PCT/US12/048319, 499/2012, PI20102724, and 2012000928) . Kauffman: Karyopharm Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Azmi: Karyopharm Therapeutics: Research Funding.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and therapy resistant malignancy. Mutant K-Ras, found in >90% of refractory PDAC, acts as a molecular switch activating Rho GTPase signaling that in turn promotes a plethora of pro-survival molecules and oncogenic microRNAs. We investigated the impact of Rho GTPase effector protein p21 activated kinase 4 (PAK4) inhibition on pro-survival p-Bad and oncogenic miRNA signaling. We demonstrate that the dual NAMPT and PAK4 modulators (KPT-9274 and KPT-9307) inhibit PDAC cell proliferation through downregulation of Bad phosphorylation and upregulation of tumor suppressive miRNAs (miR-145, let-7c, let-7d, miR-34c, miR320 and miR-100). These results suggest that targeting PAK4 could become a promising approach to restore pro-apoptotic function of Bad and simultaneously activate tumor suppressive miRNAs in therapy resistant PDAC.

Introduction: The chemoimmunotherapeutic regimen of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) is curative for many patients (pts) with diffuse large B cell lymphoma (DLBCL), but a subset of high-risk pts, including those with transformed and double-hit lymphomas, are less likely to be cured by R-CHOP. Exportin-1 (XPO1), a nuclear export protein, is overexpressed in NHL and correlates with chemotherapy-resistance and poor prognosis. Overexpression of XPO1 results in increased nuclear export of tumor suppressor proteins (TSPs) leading to their functional inactivation, and enhanced expression of Bcl-2 family members and c-Myc. Inhibition of XPO1 leads to TSP activation and oncoprotein reduction. Selinexor, a novel, oral selective inhibitor of XPO1, was approved by the FDA for DLBCL after 2 prior therapies based on an overall response rate (ORR) of 29% including a 13% complete response (CR) rate. Our previous preclinical results in vitro and in vivo showed potent additive or synergistic cytotoxicity of selinexor with chemotherapy in xenograft NHL mouse models. We evaluated the safety and preliminary efficacy of the combination of selinexor with R-CHOP chemotherapy in pts with untreated NHL. Methods: This is an open-label phase 1b study to determine the safety, tolerability, recommended phase 2 dose (RP2D) and maximum tolerated dose (MTD) of selinexor in combination with R-CHOP in pts with NHL. Pts with stage III/IV DLBCL (including transformed DLBCL) who had no prior therapy, and pts with indolent lymphomas who had either no prior therapy or received one prior therapy not containing an anthracycline, were included. A standard 3 + 3 dose escalation design in which pts received 6 cycles of R-CHOP (rituximab 375 mg/m2, cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2 IV, vincristine 0.5 mg/kg on day 1, prednisone 100 mg po on days 1-5 in a 21 day cycle) and 60 mg or 80 mg selinexor on days 1, 8, and 15 of each cycle was used. Weekly selinexor maintenance, at the dose employed at the conclusion of R-CHOP, was continued for an additional year. Blood samples were obtained on days 0, 3, 5, 8 and 15 for RNA isolation followed by RT-PCR to evaluate the expression of targets or downstream effectors including XPO1, AABCG2, AKT, Bcl-2, Bax and ERK. Results: A total of 12 pts were enrolled: 10 pts had DLBCL (1 GCB, 5 non-GCB, 4 transformed from indolent NHL); 2 pts had follicular lymphoma. Median age was 55 years. Six pts received selinexor 60 mg weekly + R-CHOP and 6 pts received selinexor 80 mg weekly + R-CHOP. Among the first 3 pts in the 60 mg cohort, there was an episode of grade 3 supraventricular tachycardia and an episode of grade 3 syncope. No further cardiac events occurred amongst these 3 pts, and this cohort was expanded by 3 pts. No further serious cardiac adverse events (AEs) occurred for the entire cohort. Among the first 3 pts in the 80 mg cohort, 1 DLT occurred: grade 3 nausea and vomiting. There were more discontinuations and dose reductions in the 80 mg cohort than in the 60 mg cohort; The majority of AEs were grade 1 or 2 and included nausea (100%), fatigue (67%), skin and nail changes (58%), constipation (42%), dizziness (42%), sinus congestion (42%), and vomiting (42%) with nausea and fatigue lasting ~1-2 days. AEs leading to selinexor discontinuation in 5 pts included fatigue +/- nausea. Dose reductions occurred in 3 pts, all due to fatigue. 8 pts completed all 6 cycles of R-CHOP with Selinexor, and 6 continued maintenance therapy. 2 pts came off study for non-compliance. MTD was not reached, however because of the higher frequency of selinexor discontinuation and dose reductions in the 80 mg cohort, selinexor 60 mg weekly + R-CHOP is the recommended RP2D. Among 10 efficacy evaluable pts (6 at 60 mg and 4 at 80 mg), the ORR was 100%: 9 pts with a CR (including 4 non-GCB pts) and 1 with a PR. Median follow up is 476 days. In line with previous reports, XPO1 mRNA expression was consistiently increased in all pt blood samples at day 3. Conclusions: R-CHOP in combination with weekly selinexor showed encouraging preliminary efficacy with an ORR of 100% and CR of 90%. At the 60 mg selinexor dose level, AEs were manageable and milder (grade 1/2) than those previously reported with selinexor. The results of correlative studies performed were consistent with our preclinical findings. A phase 2 study of this combination is ongoing in frontline DLBCL and Richter's transformation (NCT03147885). Disclosures Seymour: Incyte: Research Funding; Janssen/Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Genentech: Research Funding; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding. Ramchandren:Seattle Genetics, Sandoz-Novartis, Pharmacyclics, an AbbVie Company, Janssen, Bristol-Myers Squibb: Consultancy; Merck, Seattle Genetics, Janssen, Genentech: Research Funding. Yang:Protagonist: Research Funding; AROG: Research Funding; AstraZeneca: Research Funding; Jannsen: Research Funding. Bhutani:Sanofi: Consultancy, Research Funding. Azmi:Rhizen Phamaceuticals Inc, EISAI: Research Funding; GLC, Guidepoint: Consultancy. Zonder:Intellia, Amgen, Takeda, Janssen, Regeneron, Alnylam, Caelum, Oncopeptides: Consultancy; BMS, Celgene: Research Funding.

The current study was undertaken to investigate the effect of differentially formulated polyphenolic compound Essential Turmeric Oil-Curcumin (ETO-Cur), and Tocotrienol-rich fraction (TRF) of vitamin E isomers on colorectal cancer (CRC) cells that produce aggressive tumors. Combinations of ETO-Cur and TRF were used to determine the combinatorial effects of ETO-Cur and TRF-mediated inhibition of growth of CRC cells in vitro and HCT-116 cells xenograft in SCID mice. 16S rRNA gene sequence profiling was performed to determine the outcome of gut microbial communities in mice feces between control and ETO-Cur-TRF groups. Bacterial identifications were validated by performing SYBR-based Real Time (RT) PCR. For metagenomics analysis to characterize the microbial communities, multiple software/tools were used, including Quantitative Insights into Microbial Ecology (QIIME) processing tool. We found ETO-Cur and TRF to synergize and that the combination of ETO-Cur-TRF significantly inhibited growth of HCT-116 xenografts in SCID mice. This was associated with a marked alteration in microbial communities and increased microbial OTU (operation taxonomic unit) number. The relative abundance of taxa was increased and the level of microbial diversity after 34 days of combinatorial treatment was found to be 44% higher over the control. Shifting of microbial family composition was observed in ETO-Cur-TRF treated mice as evidenced by marked reductions in Bacteroidaceae, Ruminococcaceae, Clostridiales, Firmicutes and Parabacteroids families, compared to controls. Interestingly, during the inhibition of tumor growth in ETO-Cur treated mice, probiotic Lactobacillaceae and Bifidobacteriaceae were increased by 20-fold and 6-fold, respectively. The relative abundance of anti-inflammatory Clostridium XIVa was also increased in ETO-Cur-TRF treated mice when compared with the control. Our data suggest that ETO-Cur-TRF show synergistic effects in inhibiting colorectal cancer cell proliferation in vitro and in mouse xenografts in vivo, and might induce changes in microbial diversity in mice.

Mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS) are among the most common aberrations in cancer, including non-small cell lung cancer (NSCLC). The lack of an ideal small molecule binding pocket in KRAS protein and its high affinity towards the abundance of cellular guanosine triphosphate (GTP) renders the design of specific small molecule drugs challenging. Despite efforts, KRAS remains a challenging therapeutic target. Among the different known mutations; the KRAS(G12C) (glycine 12 to cysteine) mutation has been considered potentially druggable. Several novel covalent direct inhibitors targeting KRAS(G12C) with similar covalent binding mechanisms are now in clinical trials. Both AMG 510 from Amgen and MRTX849 from Mirati Therapeutics covalently binds to KRAS(G12C) at the cysteine at residue 12, keeping KRAS(G12C) in its inactive GDP-bound state and inhibiting KRAS-dependent signaling. Both inhibitors are being studied as single agent or as combination with immunotherapy phase 2 trials. In addition, two novel KRAS G12C inhibitors JNJ-74699157 and LY3499446 will have entered phase 1 studies by the end of 2019. Given the rapid clinical development of 4 direct covalent KRAS G12C inhibitors within a short period of time, understanding the similarities and differences among these will be important to determine the best treatment option based on tumor specific response (NSCLC versus colorectal carcinoma), potential resistance mechanisms (i.e. anticipated acquired mutation at the cysteine 12 residue) and central nervous system (CNS) activity. Additionally, further investigation evaluating the efficacy and safety of combination therapies with agents such as immune checkpoint inhibitors will be important next steps.

The disease-preventive benefits of many natural agents, particularly dietary compounds, have been attributed to their well-recognized activity as regulators of the redox state of the cell. Nevertheless, most of these studies have focused on their antioxidant activity. A large body of evidence indicates that several of these agents can elicit prooxidant behavior that has been linked to their anticancer effects. More than a decade ago, our team demonstrated the prooxidant anticancer effects of several such natural compounds. We proposed that dietary natural products, particularly plant-derived polyphenolic compounds, can take advantage of the elevated copper levels in the tumor microenvironment and promote reactive oxygen species generation, leading to cancer cell-selective DNA damage. Over the last decade, we have proven this hypothesis in cell-free, cellular, and animal tumor models. Our studies highlight an important yet underappreciated role of natural dietary compounds as prooxidants that can have therapeutic significance.

INTRODUCTION: The Ca2+release-activated Ca2+ (CRAC) channel, composed of Orai and STIM proteins, represents one of the main routes of Ca(2+) entry in most non-excitable cells. There is accumulating evidence to suggest that CRAC channel can influence various processes associated with tumorigenesis. Overexpression of CRAC channel proteins has been observed in several types of cancer tissues and cells, indicating that blocking CRAC channel activated Ca(2+) influx can have therapeutic benefits for cancer patients. AREAS COVERED: In this review, we have primarily focused on the molecular composition and activation mechanism of CRAC channel as well as the myriad roles this Ca(2+) channel play in various cancers. We further describe relevant information about several efforts aimed at developing CRAC channel blockers and their likely implications for cancer therapy. We have extensively utilized the available literature on PubMed to this end. EXPERT OPINION: The possibility of targeting CRAC channel mediated Ca(2+) entry in cancer cells has generated considerable interest in recent years. Use of CRAC channel blockers in cancer preclinical studies and clinical trials has been relatively limited as compared to other diseases. The future lies in developing and testing more potent and selective drugs that target cancer cell specific CRAC channel proteins, hence opening better avenues for cancer therapeutic development.

Lenvatinib is a multitargeted tyrosine kinase inhibitor (TKI) that shows improved median progression-free survival (PFS) in patients with thyroid carcinomas. However, virtually all patients ultimately progress, indicating the need for a better understanding of the mechanisms of resistance. Here, we examined the molecular profile of anaplastic thyroid cancer cells (8505C) exposed to lenvatinib and found that long-term exposure to lenvatinib caused phenotypic changes. Consistent with change toward mesenchymal morphology, activation of pro-survival signaling, nuclear exporter protein exportin 1 (XPO1) and Rho GTPase effector p21 activated kinases (PAK) was also observed. RNA-seq analysis showed that prolonged lenvatinib treatment caused alterations in numerous cellular pathways and several oncogenes such as CEACAM (carcinoembryonic antigen-related cell adhesion molecule) and NUPR1 (Nuclear protein 1) were also upregulated. Further, we evaluated the impact of XPO1 and PAK4 inhibition in the presence or absence of lenvatinib. Targeted inhibition of XPO1 and PAK4 could sensitize the 8505C cells to lenvatinib. Both XPO1 and PAK4 inhibitors, when combined with lenvatinib, showed superior anti-tumor activity in 8505C sub-cutaneous xenograft. These studies bring forward novel drug combinations to complement lenvatinib for treating anaplastic thyroid cancer. Such combinations may possibly reduce the chances of lenvatinib resistance in thyroid cancer patients.

Pancreatic neuroendocrine tumors (PNET) remain an unmet clinical need. In this study, we show that targeting both nicotinamide phosphoribosyltransferase (NAMPT) and p21-activated kinase 4 (PAK4) could become a synthetic lethal strategy for PNET. The expression of PAK4 and NAMPT was found to be higher in PNET tissue compared to normal cells. PAK4-NAMPT dual RNAi suppressed proliferation of PNET cell lines. Treatment with KPT-9274 (currently in a Phase I trial or analogs, PF3758309 (the PAK4 selective inhibitor) or FK866 (the NAMPT inhibitor)) suppressed the growth of PNET cell lines and synergized with the mammalian target of rapamycin (mTOR) inhibitors everolimus and INK-128. Molecular analysis of the combination treatment showed down-regulation of known everolimus resistance drivers. KPT-9274 suppressed NAD pool and ATP levels in PNET cell lines. Metabolomic profiling showed a statistically significant alteration in cellular energetic pathways. KPT-9274 given orally at 150 mg/kg 5 days/week for 4 weeks dramatically reduced PNET sub-cutaneous tumor growth. Residual tumor analysis demonstrated target engagement in vivo and recapitulated in vitro results. Our investigations demonstrate that PAK4 and NAMPT are two viable therapeutic targets in the difficult to treat PNET that warrant further clinical investigation.

Gastric cancer remains an unmet clinical problem in urgent need of newer and effective treatments. Here we show that the nuclear export protein, Exportin 1 (XPO1, chromosome region maintenance 1 or CRM1), is a promising molecular target in gastric cancer. We demonstrate significant overexpression of XPO1 in a cohort of histologically diverse gastric cancer patients with primary and metastatic disease. XPO1 RNA interference suppressed gastric cancer cell growth. Anti-tumor activity was observed with specific inhibitor of nuclear export (SINE) compounds (selinexor/XPOVIO), second-generation compound KPT-8602/eltanexor, KPT-185 and +ve control Leptomycin B in three distinct gastric cancer cell lines. SINE compounds inhibited gastric cancer cell proliferation, disrupted spheroid formation, induced apoptosis and halted cell cycle progression at the G1/S phase. Anti-tumor activity was concurrent with nuclear retention of tumor suppressor proteins and inhibition of colony formation. In combination studies, SINE compounds enhanced the efficacy of nab-paclitaxel in vitro and in vivo. More significantly, using non-coding RNA sequencing studies, we demonstrate for the first time that SINE compounds can alter the expression of non-coding RNAs (microRNAs and piwiRNAs). SINE treatment caused statistically significant downregulation of oncogenic miR-33b-3p in two distinct cell lines. These studies demonstrate the therapeutic significance of XPO1 in gastric cancer that warrants further clinical investigation.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) remains a deadly disease urgently requiring new treatments. Overexpression of the protein transporter exportin-1 (XPO1) leads to mislocalization of tumor-suppressor proteins (TSP) and their inactivation. Earlier, we showed that blocking XPO1 by CRISPR/Cas9 validated Selective Inhibitor of Nuclear Export (SINE) compounds (selinexor and analogs) restores the antitumor activity of multiple TSPs leading to suppression of PDAC in vitro and in orthotopic models. Experimental Design: We evaluate the synergy between SINE compounds and standard-of-care treatments in preclinical models and in a PDAC Phase Ib trial. Results: SINE compounds synergize with gemcitabine (GEM) and nanoparticle albumin–bound (nab)-paclitaxel leading to suppression of PDAC cellular growth and cancer stem cell (CSC) spheroids disintegration. Label-free quantitative proteome profiling with nuclear and cytoplasmic enrichment showed superior enhancement in nuclear protein fraction in combination treatment. Selinexor inhibited the growth of PDAC CSC and two patient-derived (PDX) subcutaneous xenografts. Selinexor–GEM–nab-paclitaxel blocked PDX and orthotopic tumor growth. In a phase 1b study (NCT02178436), 9 patients were exposed to selinexor (60 mg oral) with GEM (1,000 mg/m2 i.v.) and nab-paclitaxel (125 mg/m2 i.v.) on days 1, 8, and 15 of 28-day cycle. Two patients showed partial response, and 2 had stable disease. An outstanding, durable objective response was observed in one of the responders with progression-free survival of 16 months and overall survival of 22 months. Conclusions: Our preclinical and ongoing clinical study lends support to the use of selinexor–GEM–nab-paclitaxel as an effective therapy for metastatic PDAC.

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.

The global incidence of gastric cancer exceeds 1 million people per year and has a 5-year survival rate of 31%. Current chemotherapies are not effective because of genetic and epigenetic heterogeneity and late stage detection. Previously, we showed that exportin 1 (XPO1) is a therapeutic target in gastric cancer, as it facilitates the nuclear export of various cancer related tumor suppressor proteins (TSPs) through direct interaction with their nuclear export sequences (NES). XPO1 is upregulated in a variety of tumors including gastric cancer, leading to increased cytoplasmic localization of TSPs supporting malignant transformation and cancer cell proliferation, Knockdown of XPO1 leads to anti-tumor effects and pharmacologic targeting of XPO1 is achievable with selective inhibitors of nuclear export (SINE) compounds. In 2019 and 2020, the first generation XPO1 inhibitor KPT-330 was FDA approved for the use in penta-refractory multiple myeloma and non-Hodgkin's Lymphoma. Besides protein, mRNA and non-coding RNAs are also transported across the nuclear membrane. Non-coding RNA transport is generally attributed to another nuclear export protein, exportin 5 (XPO5), which plays a critical role in microRNA (miRNA) biogenesis. Nevertheless, there is emerging evidence supporting the role of XPO1 in non-coding RNA transport. In order to elucidate the role of XPO1 in non-coding RNA biology, we performed small RNA sequencing on gastric cancer cell lines treated with the XPO1 inhibitor selinexor. Our results revealed statistically significant changes in miR-7974 and miR-129-1-3p. These two miRs are recognized to influence protein modifications and cell survival. Administration of a miR-7974 mimic and a miR-129-1-3p inhibitor reduced proliferation of gastric cancer cells. Next, we evaluated the miR target genes of interest. At the mRNA level, we found upregulation of RasGEF Domain Family Member 1a (RASGEF1A), downregulation of DNA damage-regulated autophagy modulator 1 (DRAM1), downregulation of TIMP2 and downregulation of Cyclin G2 protein encoding gene CCNG2. Western Blots confirmed inhibition of AKT, RAS and Wnt signaling. Our results also showed that miR-7974 overexpression correlated with cellular XPO1 levels through the enhancement of XPO1 and its cargoes RCC1 and RANBP2. Finally inhibition of XPO1 by SINE compounds reversed the observed oncogenic phenotypes caused by these microRNAs. Our results unveil a novel interaction network between XPO1 and small non-coding RNAs and show that XPO1 induced small-noncoding microRNAs influence gastric cancer proliferation, and can be reversed by SINE compounds. This implies that modulation of microRNAs might be explored as a biomarker for XPO1 inhibitor therapy and warrants further investigation. Citation Format: Rachel E. Sexton, Mohammad Najeeb Al-Hallak, Bayan Al-Share, Yiwei Li, Amro Aboukameel, Yosef Landesman, Trinayan Kashyap, Steve Kim, Ramzi M. Mohammad, Philip A. Philip, Asfar S. Azmi. Mir-7974: An oncogenic miRNA that perpetuates gastric cancer [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 2383.

Aberrant nuclear protein transport, often observed in cancer, causes mislocalization-dependent inactivation of critical cellular proteins. Earlier we showed that overexpression of exportin 1 is linked to higher grade and Gleason score in metastatic castration resistant prostate cancer (mCRPC). We also showed that a selective inhibitor of nuclear export (SINE) selinexor and second generation eltanexor (KPT-8602) could suppress mCRPC growth, reduce androgen receptor (AR), and re-sensitize to androgen deprivation therapy. Here we evaluated the combination of KPT-8602 with PARP inhibitors (PARPi) olaparib, veliparib and rucaparib in 22rv1 mCRPC cells. KPT-8602 synergized with PARPi (CI &lt, 1) at pharmacologically relevant concentrations. KPT-8602-PARPi showed superior induction of apoptosis compared to single agent treatment and caused up-regulation of pro-apoptotic genes BAX, TP53 and CASPASE 9. Mechanistically, KPT-8602-PARPi suppressed AR, ARv7, PSA and AR targets FOXA1 and UBE2C. Western blot analysis revealed significant down-regulation of AR, ARv7, UBE2C, SAM68, FOXA1 and upregulation of cleaved PARP and cleaved CASPASE 3. KPT-8602 with or without olaparib was shown to reduce homologous recombination-regulated DNA damage response targets including BRCA1, BRCA2, CHEK1, EXO1, BLM, RAD51, LIG1, XRCC3 and RMI2. Taken together, this study revealed the therapeutic potential of a novel combination of KPT-8602 and PARP inhibitors for the treatment of mCRPC.

e20533 Background: Nuclear protein transport is essential in guiding the organized traffic of important proteins and RNAs between the nucleus and cytoplasm of the cell. Export of proteins from the nucleus is exclusively regulated by Exportin 1(XPO1). In cancer, XPO1 is universally hyperactive and can promote the export of important tumor suppressors to the cytoplasm, leading to their functional inactivation. XPO1 is aberrantly over expressed in NSCLC and this over expression has been linked to poor overall survival. The underlying mechanisms of XPO1 over expression are not known. Currently there are no studies evaluating the impact of XPO1 mutations on NSCLC incidence and therapy resistance. Additionally, there are no studies that examined the XPO1 related pathways in NSCLC harboring co-alterations with other driver mutations such as EGFR or ALK. Methods: Tumor samples were analyzed using next-generation sequencing (NextSeq, 592 Genes), IHC, and whole transcriptome sequencing (WTS ,NovaSeq) (Caris Life Sciences, Phoenix, AZ). PD-L1 expression was tested by IHC using 22c3 (Dako) and TPS scores were reported (cutoff > 1%). TMB was measured by totaling somatic mutations (TMB-high cut-off ³ 10 mutations per Megabase). Gene fusions were detected by RNA sequencing using either the Archer FusionPlex panel or WTS. Survival was extracted from insurance claims data and calculated from time of tissue collection to last contact using Kaplan-Meier estimate. Statistical significance was determined using chi-square and Wilcoxon rank sum test and adjusted for multiple comparisons. Results: Among 18,218 NSCLC tumors sequenced, 26 harbored XPO1 mutations. XPO1 mutant tumors were more likely to be TMB High(79% vs. 52%, p = 0.007) and less likely to have high PDL1(32% vs. 68%, p = 0.03). KRAS mutations were seen in 19%(n = 5), EGFR mutation were rare (n = 2), and no targetable fusions were seen. Among the 17,449 NSCLC tumors with clinical data, there were 24 XPO1 mutant tumors with no histology imbalance observed in mutant vs. wild-type(WT). Comparison of survival in the NSCLC group between XPO1 mutant and WT showed a negative association with a hazard ratio(HR) of 1.932 (95% CI: 1.144- 3.264 p = 0.012). Comparing the survival within the subgroup with confirmed adenocarcinoma histology (9973 XPO1 WT and 14 XPO1 mutant) showed a similar negative correlation in survival with a HR of 2.156 (95% CI: 1.027- 4.525 P = 0.037). Conclusions: Presence of XPO1 pathogenic mutations was associated with a poor survival in both the entire NSCLC cohort and the adenocarcinoma subgroup. Further studies of this negative association at the molecular level along with effect of other co-existing mutations can result in development of novel treatment strategies.

The p21-activated kinase 4 (PAK4) is a key downstream effector of the Rho family GTPases and is found to be overexpressed in pancreatic ductal adenocarcinoma (PDAC) cells but not in normal human pancreatic ductal epithelia (HPDE). Gene copy number amplification studies in PDAC patient cohorts confirmed PAK4 amplification making it an attractive therapeutic target in PDAC. We investigated the antitumor activity of novel PAK4 allosteric modulators (PAM) on a panel of PDAC cell lines and chemotherapy-resistant flow-sorted PDAC cancer stem cells (CSC). The toxicity and efficacy of PAMs were evaluated in multiple subcutaneous mouse models of PDAC. PAMs (KPT-7523, KPT-7189, KPT-8752, KPT-9307, and KPT-9274) show antiproliferative activity in vitro against different PDAC cell lines while sparing normal HPDE. Cell growth inhibition was concurrent with apoptosis induction and suppression of colony formation in PDAC. PAMs inhibited proliferation and antiapoptotic signals downstream of PAK4. Co-immunoprecipitation experiments showed disruption of PAK4 complexes containing vimentin. PAMs disrupted CSC spheroid formation through suppression of PAK4. Moreover, PAMs synergize with gemcitabine and oxaliplatin in vitro. KPT-9274, currently in a phase I clinical trial (clinicaltrials.gov; NCT02702492), possesses desirable pharmacokinetic properties and is well tolerated in mice with the absence of any signs of toxicity when 200 mg/kg daily is administered either intravenously or orally. KPT-9274 as a single agent showed remarkable antitumor activity in subcutaneous xenograft models of PDAC cell lines and CSCs. These proof-of-concept studies demonstrated the antiproliferative effects of novel PAMs in PDAC and warrant further clinical investigations. Mol Cancer Ther; 16(1); 76–87. ©2016 AACR.

In previous studies we demonstrated that targeting the nuclear exporter protein exportin-1 (CRM1/XPO1) by a selective inhibitor of nuclear export (SINE) compound is a viable therapeutic strategy against Non-Hodgkin Lymphoma (NHL). Our studies along with pre-clinical work from others led to the evaluation of the lead SINE compound, selinexor, in a phase 1 trial in patients with CLL or NHL ( NCT02303392 ). Continuing our previous work, we studied combinations of selinexor-dexamethasone (DEX) and selinexor-everolimus (EVER) in NHL. Combination of selinexor with DEX or EVER resulted in enhanced cytotoxicity in WSU-DLCL2 and WSU-FSCCL cells which was consistent with enhanced apoptosis. Molecular analysis showed enhancement in the activation of apoptotic signaling and down-regulation of XPO1. This enhancement is consistent with the mechanism of action of these drugs in that both selinexor and DEX antagonize NF-κB (p65) and mTOR (EVER target) is an XPO1 cargo protein. SINE compounds, KPT-251 and KPT-276, showed activities similar to CHOP (cyclophosphamide–hydroxydaunorubicin–oncovin–prednisone) regimen in subcutaneous and disseminated NHL xenograft models in vivo. In both animal models the anti-lymphoma activity of selinexor is enhanced through combination with DEX or EVER. The in vivo activity of selinexor and related SINE compounds relative to ‘standard of care’ treatment is consistent with the objective responses observed in Phase I NHL patients treated with selinexor. Our pre-clinical data provide a rational basis for testing these combinations in Phase II NHL trials.

Background: Dihydroorotate dehydrogenase (DHODH) is an enzyme which is critically involved in process of de novo pyrimidine biosynthesis. Therefore, it plays important roles in cell proliferation, mitosis and cellular metabolism. Importantly, overexpression of DHODH has been found in various types of malignant tumors including melanoma, myeloma, and lymphoma. Moreover, from Oncomine database, we observed that colorectal and kidney cancers and lymphoma have much higher expression of DHODH compared to normal cells. These findings suggest that DHODH could be a promising target for cancer therapy. Recently, we have designed and synthesized a novel small molecule inhibitor (RP7214) of DHODH and found that it has potent inhibitory effects in acute myeloid leukemia. However, the inhibitory effect of RP7214 on solid tumor-derived cell lines and the downstream oncogenic markers is not completely understood. Methods: In vitro studies aimed at elucidating the effects of RP7214 on cell proliferation and the underlying molecular mechanisms were conducted using breast adenocarcinoma (MDA-MB-231) and colorectal carcinoma cell lines (HT-29 and HCT-116). We also investigated whether RP7214 could potentiate the anti-cancer activities of proven chemotherapeutics such as Verzenio, Doxorubicin, and Oxaliplatin, for the treatment of breast and colorectal cancers. Results: RP7214 significantly (P Conclusions: Based on these results, we conclude that RP7214 could be a promising DHODH inhibitor used in combination with conventional chemotherapeutics for better treatment of breast and colorectal cancers. We are now conducting animal experiment to confirm the value of RP7214 in vivo in combination treatment of breast and colorectal cancers. Citation Format: Asfar S. Azmi, Amro Aboukameel, Mohammad Najeeb Al-Hallak, Philip A. Philip, Kumar Penmetsa, Srikant Visvanadha, Gabriel Mpilla, Rachel Sexton, Ramzi M. Mohammad. A novel, small molecule inhibitor of dihydroorotate dehydrogenase (DHODH), RP7214, potentiates activity of chemotherapeutics in breast and colorectal cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 234.

Pancreatic ductal adenocarcinoma (PDAC) is the third-highest cause of cancer-related death in the United States. The heterogenicity of the PDAC is a major challenge in the success of anti-PDAC therapy. PDAC is comprised of >90 % dense desmoplastic tumor stroma that impedes the delivery of drugs into the core of the tumor, resulting development of drug resistance. Thus, we developed a nanoparticle pro-drug to overcome the stromal barrier of PDAC. We utilized nanoengineering to target dual CD44 and c-Met biomarkers that have a significant role in developing drug resistance on PDAC. Western blot analysis in various PDAC cell lines, such as PANC-1, AsPc-1, and BxPc3 revealed a high abundance of CD44 and cMet, strongly supporting our hypothesis. Therefore, targeting the CD44 and c-Met biomarker and selectively delivering anti-PDAC drug is a smart strategy to improve the anti-PDAC outcome. We developed dual CD44/c-Met targeted nanoparticle, called D44M that can efficiently penetrate the core of tumor as well as home into CD44 and cMet biomarker. We evaluated tumor selectivity of the near infrared (NIR) fluorescent dye conjugated D44M, D44M-NIR in the orthotopic PDAC model. The biodistribution of D44M-NIR in orthotopic animal model proved superior tumor penetration of D44M-NIR with 7-fold higher than the control NIR dye treatment. Moreover, D44M-NIR treated showed minimum liver accumulation, suggesting an outstanding approach of overcoming nanoparticle mediated liver toxicity. Finally, the D44M was chemically conjugated with gemcitabine (GEM), namely D44M-GEM pro-drug that showed excellent PDAC killing. The PDAC cell killing study of D44M-GEM in combination with Everolimus revealed high synergistic combination effect with CI value of 0.2-0.5. The anti-PDAC therapy in ASPC-1 xenograft model demonstrated significant tumor growth inhibition for treatment of D44M-GEM and D44M-GEM+Everolimus as compared to control. Supported with data, our work provides a perspective towards the implementation of nanotherapeutic to overcome the bottleneck barrier of therapeutic delivery and efficacy of PDAC management. Citation Format: Rami Alzhrani, Samaresh Sau, Amro Aboukameel, Hashem Alsaab, Asfar Azmi, Arun Iyer. Nanoparticle pro-drug to overcome the stromal barrier in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1722.

The high attrition rates in cancer drug discovery indicate that there is a need to revamp the ways in which precursor preclinical research is being performed. The majority of preclinical studies are either done in cancer cell lines (in 2D or 3D culture) or subcutaneous xenograft. Recently, patient derived xenograft (PDX) and patient organoid derived (PDOx) mice tumor models have emerged. However, these preclinical models are not truly reflective of the tumor heterogeneity and microenvironment. PDX are usually grown subcutaneously and are infiltrated with mouse stroma. The orthotopic models are developed by injecting an organoid suspension at the organ site. Unfortunately, the organoid models do not recapitulate the tumor mass which is composed of so many different types of closely knit cells (fibroblast, stellate, tumor cells, immune cells among others). Genetically engineered models are more representative of human tumors and recapitulate the microenvironment to a significant extent. Given they are developed in immune-competent mice, therefore, they also are able to help in immune-related cancer biology and drug discovery studies (though mice immune system). Although cumbersome and costly, genetic mice models serve as better models for cancer development and drug discovery studies. In this chapter, an attempt has been made to provide an overview of the protocols in genetic mice breeding with a focus on pancreatic cancer genetically engineered models.