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Background Ulixertinib is a novel oral ERK inhibitor that has shown promising single-agent activity in a phase I clinical trial that included patients with RAS-mutant cancers. Methods We conducted a phase Ib trial combining ulixertinib with gemcitabine and nab-paclitaxel (GnP) for untreated metastatic pancreatic adenocarcinoma. The trial comprised a dose de-escalation part and a cohort expansion part at the recommended phase II dose (RP2D). Primary endpoint was to determine the RP2D of ulixertinib plus GnP and secondary endpoints were to assess toxicity and safety profile, biochemical and radiographic response, progression-free survival (PFS) and overall survival (OS). Results Eighteen patients were enrolled. Ulixertinib 600 mg PO twice daily (BID) with GnP was initially administered but was de-escalated to 450 mg BID as RP2D early during dose expansion due to poor tolerability, which ultimately led to premature termination of the study. Common treatment-related adverse events (TRAEs) were anemia, thrombocytopenia, rash and diarrhea. For 5 response evaluable patients, one patient achieved a partial response and 2 patients achieved stable disease. For 15 patients who received the triplet, median PFS and OS were 5.46 and 12.23 months, respectively. Conclusion Ulixertinib plus GnP had similar frequency of grade ≥3 TRAEs and potentially efficacy as GnP, however was complicated by a high rate of all-grade TRAEs (ClinicalTrials.gov Identifier: NCT02608229).

Purpose: To assess the preclinical efficacy, clinical safety and efficacy, and MTD of palbociclib plus nab-paclitaxel in patients with advanced pancreatic ductal adenocarcinoma (PDAC). Experimental Design: Preclinical activity was tested in patient-derived xenograft (PDX) models of PDAC. In the open-label, phase I clinical study, the dose-escalation cohort received oral palbociclib initially at 75 mg/day (range, 50‒125 mg/day; modified 3+3 design; 3/1 schedule); intravenous nab-paclitaxel was administered weekly for 3 weeks/28-day cycle at 100‒125 mg/m2. The modified dose–regimen cohorts received palbociclib 75 mg/day (3/1 schedule or continuously) plus nab-paclitaxel (biweekly 125 or 100 mg/m2, respectively). The prespecified efficacy threshold was 12-month survival probability of ≥65% at the MTD. Results: Palbociclib plus nab-paclitaxel was more effective than gemcitabine plus nab-paclitaxel in three of four PDX models tested; the combination was not inferior to paclitaxel plus gemcitabine. In the clinical trial, 76 patients (80% received prior treatment for advanced disease) were enrolled. Four dose-limiting toxicities were observed [mucositis (n = 1), neutropenia (n = 2), febrile neutropenia (n = 1)]. The MTD was palbociclib 100 mg for 21 of every 28 days and nab-paclitaxel 125 mg/m2 weekly for 3 weeks in a 28-day cycle. Among all patients, the most common all-causality any-grade adverse events were neutropenia (76.3%), asthenia/fatigue (52.6%), nausea (42.1%), and anemia (40.8%). At the MTD (n = 27), the 12-month survival probability was 50% (95% confidence interval, 29.9–67.2). Conclusions: This study showed the tolerability and antitumor activity of palbociclib plus nab-paclitaxel treatment in patients with PDAC; however, the prespecified efficacy threshold was not met. Trial Registration: Pfizer Inc (NCT02501902) Significance: In this article, the combination of palbociclib, a CDK4/6 inhibitor, and nab-paclitaxel in advanced pancreatic cancer evaluates an important drug combination using translational science. In addition, the work presented combines preclinical and clinical data along with pharmacokinetic and pharmacodynamic assessments to find alternative treatments for this patient population.

Neoantigen burden and CD8 T cell infiltrate are associated with clinical outcome in pancreatic ductal adenocarcinoma (PDAC). A shortcoming of many genetic models of PDAC is the lack of neoantigen burden and limited T cell infiltrate. The goal of the present study was to develop clinically relevant models of PDAC by inducing cancer neoantigens in KP2, a cell line derived from the KPC model of PDAC. KP2 was treated with oxaliplatin and olaparib (OXPARPi), and a resistant cell line was subsequently cloned to generate multiple genetically distinct cell lines (KP2-OXPARPi clones). Clones A and E are sensitive to immune checkpoint inhibition (ICI), exhibit relatively high T cell infiltration, and have significant upregulation of genes involved in antigen presentation, T cell differentiation, and chemokine signaling pathways. Clone B is resistant to ICI and is similar to the parental KP2 cell line in terms of relatively low T cell infiltration and no upregulation of genes involved in the pathways noted above. Tumor/normal exome sequencing and in silico neoantigen prediction confirms successful generation of cancer neoantigens in the KP2-OXPARPi clones and the relative lack of cancer neoantigens in the parental KP2 cell line. Neoantigen vaccine experiments demonstrate that a subset of candidate neoantigens are immunogenic and neoantigen synthetic long peptide vaccines can restrain Clone E tumor growth. Compared to existing models, the KP2-OXPARPi clones better capture the diverse immunobiology of human PDAC and may serve as models for future investigations in cancer immunotherapies and strategies targeting cancer neoantigens in PDAC.

Numerous cell states are known to comprise the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the developmental stemness and co-occurrence of these cell states remain poorly defined. Here, we performed single-cell RNA sequencing (scRNA-seq) on a cohort of treatment-naive PDAC time-of-diagnosis endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) samples (n=25) and surgical resections (n=6), which were then combined with publicly available samples to increase statistical power (n=80). Following annotation into 25 distinct cell states, cells were scored for developmental stemness and a customized version of the Ecotyper tool was used to identify communities of co-occurring cell states in bulk RNA-seq samples (n=268). We discovered a tumor microenvironmental community comprised of aggressive basal-like malignant cells, tumor-promoting SPP1+ macrophages, and myofibroblastic cancer-associated fibroblasts associated with especially poor prognosis. We also found a developmental stemness continuum with implications for survival that is present in both malignant cells and cancer-associated fibroblasts (CAFs). We further demonstrated that high-dimensional analyses predictive of survival are feasible using standard-of-care, time-of-diagnosis EUS-FNB specimens. In summary, we identified novel tumor microenvironmental and developmental stemness characteristics from a high-dimensional gene expression analysis of PDAC using human tissue specimens including time-of-diagnosis EUS-FNB samples. These reveal new connections between tumor microenvironmental composition, CAF and malignant cell stemness, and patient survival that could lead to better upfront risk stratification and more personalized upfront clinical decision-making.

Purpose:To assess the preclinical efficacy, clinical safety and efficacy, and MTD of palbociclib plus nab-paclitaxel in patients with advanced pancreatic ductal adenocarcinoma (PDAC).Experimental Design:Preclinical activity was tested in patient-derived xenograft (PDX) models of PDAC. In the open-label, phase I clinical study, the dose-escalation cohort received oral palbociclib initially at 75 mg/day (range, 50‒125 mg/day; modified 3+3 design; 3/1 schedule); intravenous nab-paclitaxel was administered weekly for 3 weeks/28-day cycle at 100‒125 mg/m2. The modified dose–regimen cohorts received palbociclib 75 mg/day (3/1 schedule or continuously) plus nab-paclitaxel (biweekly 125 or 100 mg/m2, respectively). The prespecified efficacy threshold was 12-month survival probability of ≥65% at the MTD.Results:Palbociclib plus nab-paclitaxel was more effective than gemcitabine plus nab-paclitaxel in three of four PDX models tested; the combination was not inferior to paclitaxel plus gemcitabine. In the clinical trial, 76 patients (80% received prior treatment for advanced disease) were enrolled. Four dose-limiting toxicities were observed [mucositis (n = 1), neutropenia (n = 2), febrile neutropenia (n = 1)]. The MTD was palbociclib 100 mg for 21 of every 28 days and nab-paclitaxel 125 mg/m2 weekly for 3 weeks in a 28-day cycle. Among all patients, the most common all-causality any-grade adverse events were neutropenia (76.3%), asthenia/fatigue (52.6%), nausea (42.1%), and anemia (40.8%). At the MTD (n = 27), the 12-month survival probability was 50% (95% confidence interval, 29.9–67.2).Conclusions:This study showed the tolerability and antitumor activity of palbociclib plus nab-paclitaxel treatment in patients with PDAC; however, the prespecified efficacy threshold was not met.Trial Registration:Pfizer Inc (NCT02501902)Significance:In this article, the combination of palbociclib, a CDK4/6 inhibitor, and nab-paclitaxel in advanced pancreatic cancer evaluates an important drug combination using translational science. In addition, the work presented combines preclinical and clinical data along with pharmacokinetic and pharmacodynamic assessments to find alternative treatments for this patient population.

Supplementary Materials and Methods

Supplementary Table S3 from A Preclinical and Phase Ib Study of Palbociclib plus Nab-Paclitaxel in Patients with Metastatic Adenocarcinoma of the Pancreas

Effective treatment for pancreatic ductal adenocarcinoma (PDAC) is an urgent, unmet medical need. Targeting KRAS, the oncogene that is present in >95% of PDAC, is a heavily pursued strategy, but remains unsuccessful in the clinic. Therefore, targeting key effector cascades of KRAS oncoprotein, particularly the mitogenic RAF–MEK–ERK pathway, represents the next best strategy. However, RAF or MEK inhibitors have failed to show clinical efficacy in PDAC. Several studies have shown that cancer cells treated with RAF or MEK inhibitors adopt multiple mechanisms to reactivate ERK signaling. Therefore, development of ERK-specific inhibitors carries the promise to effectively abrogate this pathway. Ulixertinib (or BVD-523) is a first-in-class ERK-specific inhibitor that has demonstrated promising antitumor activity in a phase I clinical trial for advanced solid tumors with NRAS and BRAF mutations, providing a strong rationale to test this inhibitor in PDAC. In this study, we show that ulixertinib effectively inhibits in vitro growth of multiple PDAC lines and potentiates the cytotoxic effect of gemcitabine. Moreover, we found that PDAC cells treated with ulixertinib upregulates the parallel PI3K–AKT pathway through activating the HER/ErbB family proteins. Concurrent inhibition of PI3K or HER proteins synergizes with ulixertinib in suppressing PDAC cell growth in vitro and in vivo. Overall, our study provides the preclinical rationale for testing combinations of ulixertinib with chemotherapy or PI3K and HER inhibitors in PDAC patients. Mol Cancer Ther; 17(10); 2144–55. ©2018 AACR.

Supplementary Figure 1. Ulixertinib potentiates gemcitabine effect and this combination is well tolerated in mice; Supplementary Figure 2. Pharmacologic ERK inhibition increases p-MEK, ERK and AKT; Supplementary Figure 3. Ulixertinib synergizes with HER inhibitors; Supplementary Figure 4. Ulixertinib synergizes with PI3K Inhibitor

Purpose:Targeting focal adhesion kinase (FAK) renders checkpoint immunotherapy effective in pancreatic ductal adenocarcinoma (PDAC) mouse model. Defactinib is a highly potent oral FAK inhibitor that has a tolerable safety profile.Patients and Methods:We conducted a multicenter, open-label, phase I study with dose escalation and expansion phases. In dose escalation, patients with refractory solid tumors were treated at five escalating dose levels of defactinib and gemcitabine to identify a recommended phase II dose (RP2D). In expansion phase, patients with metastatic PDAC who progressed on frontline treatment (refractory cohort) or had stable disease (SD) after at least 4 months of standard gemcitabine/nab-paclitaxel (maintenance cohort) were treated at RP2D. Pre- and posttreatment tumor biopsies were performed to evaluate tumor immunity.Results:The triple drug combination was well-tolerated, with no dose-limiting toxicities. Among 20 treated patients with refractory PDAC, the disease control rate (DCR) was 80%, with one partial response (PR) and 15 SDs, and the median progression-free survival (PFS) and overall survival (OS) were 3.6 and 7.8 months, respectively. Among 10 evaluable patients in the maintenance cohort, DCR was 70% with one PR and six SDs. Three patients with SD came off study due to treatment- or disease-related complications. The median PFS and OS on study treatment were 5.0 and 8.3 months, respectively.Conclusions:The combination of defactinib, pembrolizumab, and gemcitabine was well-tolerated and safe, had promising preliminary efficacy, and showed biomarker activity in infiltrative T lymphocytes. Efficacy of this strategy may require incorporation of more potent chemotherapy in future studies.

Supplemental Table 2 Patient demographics

Supplementary Figure 2. Requirement of IRAK1/4 for AI growth of PDAC cells.

Detailed description of materials, method; and supplementary figure legends

Supplementary Figure 3. IRAK1/4i is well-tolerated in mice and synergizes with gemcitabine in inducing apoptosis, suppressing proliferation and fibrosis of PDAC in vivo.

Supplementary Figure 1. Phosphorylation of IRAK4 is present in early-passage patient-derived cell lines and independent pancreatic tissue microarray.

Contains Supplemental Materials and Methods Supplemental Figures 1-6 Supplemental Table 1

Purpose: Aberrant activation of the NF-κB transcription factors underlies the aggressive behavior and poor outcome of pancreatic ductal adenocarcinoma (PDAC). However, clinically effective and safe NF-κB inhibitors are not yet available. Because NF-κB transcription factors can be activated by the interleukin-1 receptor-associated kinases (IRAKs) downstream of the Toll-like receptors (TLRs), but has not been explored in PDAC, we sought to investigate the role of IRAKs in the pathobiology of PDAC.Experimental Design: We examined the phosphorylation status of IRAK4 (p-IRAK4), the master regulator of TLR signaling, in PDAC cell lines, in surgical samples and commercial tissue microarray. We then performed functional studies using small-molecule IRAK1/4 inhibitor, RNA-interference, and CRISPR/Cas9n techniques to delineate the role of IRAK4 in NF-κB activity, chemoresistance, cytokine production, and growth of PDAC cells in vitro and in vivo.Results: p-IRAK4 staining was detectable in the majority of PDAC lines and about 60% of human PDAC samples. The presence of p-IRAK4 strongly correlated with phospho-NF-κB/p65 staining in PDAC samples and is predictive of postoperative relapse and poor overall survival. Inhibition of IRAK4 potently reduced NF-κB activity, anchorage-independent growth, chemoresistance, and secretion of proinflammatory cytokines from PDAC cells. Both pharmacologic suppression and genetic ablation of IRAK4 greatly abolished PDAC growth in mice and augmented the therapeutic effect of gemcitabine by promoting apoptosis, reducing tumor cell proliferation and tumor fibrosis.Conclusions: Our data established IRAK4 as a novel therapeutic target for PDAC treatment. Development of potent IRAK4 inhibitors is needed for clinical testing. Clin Cancer Res; 23(7); 1748–59. ©2016 AACR.

Targeting the desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) holds promise to augment the effect of chemotherapy, but success in the clinic has thus far been limited. Preclinical mouse models suggest that near-depletion of cancer-associated fibroblasts (CAF) carries a risk of accelerating PDAC progression, underscoring the need to concurrently target key signaling mechanisms that drive the malignant attributes of both CAF and PDAC cells. We previously reported that inhibition of IL1 receptor–associated kinase 4 (IRAK4) suppresses NFκB activity and promotes response to chemotherapy in PDAC cells. In this study, we report that CAF in PDAC tumors robustly express activated IRAK4 and NFκB. IRAK4 expression in CAF promoted NFκB activity, drove tumor fibrosis, and supported PDAC cell proliferation, survival, and chemoresistance. Cytokine array analysis of CAF and microarray analysis of PDAC cells identified IL1β as a key cytokine that activated IRAK4 in CAF. Targeting IRAK4 or IL1β rendered PDAC tumors less fibrotic and more sensitive to gemcitabine. In clinical specimens of human PDAC, high stromal IL1β expression associated strongly with poor overall survival. Together, our studies establish a tumor–stroma IL1β-IRAK4 feedforward signal that can be therapeutically disrupted to increase chemotherapeutic efficacy in PDAC.Significance: Targeting the IL1β-IRAK4 signaling pathway potentiates the effect of chemotherapy in pancreatic cancer. Cancer Res; 78(7); 1700–12. ©2018 AACR.

Supplementary Table 1. IRAK1 and/or IRAK4 knockdown sensitizes PDAC cells to chemotherapeutics.

Supplementary Figure 1 from The Cytoplasmic Deacetylase HDAC6 Is Required for Efficient Oncogenic Tumorigenesis

Histone deacetylase inhibitors (HDACI) are promising antitumor agents. Although transcriptional deregulation is thought to be the main mechanism underlying their therapeutic effects, the exact mechanism and targets by which HDACIs achieve their antitumor effects remain poorly understood. It is not known whether any of the HDAC members support robust tumor growth. In this report, we show that HDAC6, a cytoplasmic-localized and cytoskeleton-associated deacetylase, is required for efficient oncogenic transformation and tumor formation. We found that HDAC6 expression is induced upon oncogenic Ras transformation. Fibroblasts deficient in HDAC6 are more resistant to both oncogenic Ras and ErbB2-dependent transformation, indicating a critical role for HDAC6 in oncogene-induced transformation. Supporting this hypothesis, inactivation of HDAC6 in several cancer cell lines reduces anchorage-independent growth and the ability to form tumors in mice. The loss of anchorage-independent growth is associated with increased anoikis and defects in AKT and extracellular signal-regulated kinase activation upon loss of adhesion. Lastly, HDAC6-null mice are more resistant to chemical carcinogen-induced skin tumors. Our results provide the first experimental evidence that a specific HDAC member is required for efficient oncogenic transformation and indicate that HDAC6 is an important component underlying the antitumor effects of HDACIs. [Cancer Res 2008;68(18):7561–9]

Tumor-associated macrophages (TAMs) are abundant in pancreatic ductal adenocarcinomas (PDACs). While TAMs are known to proliferate in cancer tissues, the impact of this on macrophage phenotype and disease progression is poorly understood. We showed that in PDAC, proliferation of TAMs could be driven by colony stimulating factor-1 (CSF1) produced by cancer-associated fibroblasts. CSF1 induced high levels of p21 in macrophages, which regulated both TAM proliferation and phenotype. TAMs in human and mouse PDACs with high levels of p21 had more inflammatory and immunosuppressive phenotypes. p21 expression in TAMs was induced by both stromal interaction and/or chemotherapy treatment. Finally, by modeling p21 expression levels in TAMs, we found that p21-driven macrophage immunosuppression in vivo drove tumor progression. Serendipitously, the same p21-driven pathways that drive tumor progression also drove response to CD40 agonist. These data suggest that stromal or therapy-induced regulation of cell cycle machinery can regulate both macrophage-mediated immune suppression and susceptibility to innate immunotherapy.

Aim Aurora kinase A (AURKA) is a pleiotropic serine/threonine kinase that orchestrates mitotic progression. Paclitaxel stabilises microtubules and disrupts mitotic spindle assembly. The combination of AURKA inhibitor (alisertib) plus paclitaxel may be synergistic in rapidly proliferative cancers. We evaluated the safety and maximum tolerated dose (MTD) of alisertib in combination with nab-paclitaxel and its preliminary efficacy in patients with refractory high-grade neuroendocrine tumours (NETs). Method This is a two-part, Phase 1 study. In Part A (dose escalation), a standard 3 + 3 design was used to determine MTD. In Part B (dose expansion), patients with predominantly refractory high-grade NETs were enrolled. Results In total, 31 patients were enrolled and treated (16 in Part A and 15 in Part B). The MTD of alisertib was 40 mg BID on D1-3 per week and nab-paclitaxel 100mg/m2 weekly: 3 weeks, 1 week off. Dose-limiting toxicity was neutropenia, and other common side-effects included fatigue, mucositis, and diarrhoea. In Part A, a patient with small-cell lung cancer with partial response (PR) was treated for more than 2 years, whereas four other patients with pancreatic ductal adenocarcinoma (one patient), small cell lung cancer (SCLC) (two patients), or high-grade NET (one patient) achieved stable disease (SD). In Part B, 13 of 15 enrolled patients had high-grade NETs. Of these, one had PR, and four had SD for more than 10 months. Conclusions The combination of alisertib and nab-paclitaxel has manageable side-effect profile and showed promising preliminary efficacy in high-grade NETs, warranting further testing. Trial registration ClinicalTrials.gov identifier: NCT01677559 .

Development of candidate cancer treatments is a resource-intensive process, with the research community continuing to investigate options beyond static genomic characterization. Toward this goal, we have established the genomic landscapes of 536 patient-derived xenograft (PDX) models across 25 cancer types, together with mutation, copy number, fusion, transcriptomic profiles, and NCI-MATCH arms. Compared with human tumors, PDXs typically have higher purity and fit to investigate dynamic driver events and molecular properties via multiple time points from same case PDXs. Here, we report on dynamic genomic landscapes and pharmacogenomic associations, including associations between activating oncogenic events and drugs, correlations between whole-genome duplications and subclone events, and the potential PDX models for NCI-MATCH trials. Lastly, we provide a web portal having comprehensive pan-cancer PDX genomic profiles and source code to facilitate identification of more druggable events and further insights into PDXs’ recapitulation of human tumors., Patient-derived xenograft models (PDX) have been extensively used to study the molecular and clinical features of cancers. Here the authors present a cohort of 536 PDX models from 25 cancers, as well as their genomic and evolutionary profiles and their suitability for clinical trials.

Pancreatic ductal adenocarcinoma (PDAC) is projected to emerge as the second leading cause of cancer-related death after 2030. Extreme treatment resistance is perhaps the most significant factor that underlies the poor prognosis of PDAC. To date, combination chemotherapy remains the mainstay of treatment for most PDAC patients. Compared to other cancer types, treatment response of PDAC tumors to similar chemotherapy regimens is clearly much lower and shorter-lived. Aside from typically harboring genetic alterations that to date remain un-druggable and are drivers of treatment resistance, PDAC tumors are uniquely characterized by a densely fibrotic stroma that has well-established roles in promoting cancer progression and treatment resistance. However, emerging evidence also suggests that indiscriminate targeting and near complete depletion of stroma may promote PDAC aggressiveness and lead to detrimental outcomes. These conflicting results undoubtedly warrant the need for a more in-depth understanding of the heterogeneity of tumor stroma in order to develop modulatory strategies in favor of tumor suppression. The advent of novel techniques including single cell RNA sequencing and multiplex immunohistochemistry have further illuminated the complex heterogeneity of tumor cells, stromal fibroblasts, and immune cells. This new knowledge is instrumental for development of more refined therapeutic strategies that can ultimately defeat this disease. Here, we provide a concise review on lessons learned from past stroma-targeting strategies, new challenges revealed from recent preclinical and clinical studies, as well as new prospects in the treatment of PDAC.

Targeting the oncogenic KRAS-RAF-MEK-ERK (MAPK) pathway has remain unsuccessful in the clinic for patients with pancreatic ductal adenocarcinoma (PDAC), a highly lethal cancer with few effective treatment options. To identify novel, effective therapeutic strategies, we investigated the impact of MEK and ERK inhibitors on production of inflammatory cytokines in PDAC cells. We found that both MEK and ERK inhibitors dramatically upregulated production of TNF, leading to activation of multiple TNF signaling pathways which include the pro-death and pro-survival NF-kB and p38.MK2 cascades. Silencing of TNF receptor 1 (TNFR1) abrogated the pro-apoptotic effect of MAPK inhibitors, suggesting that pro-death effect driven by TNF signaling is required for the therapeutic effect of MAPK inhibitors. However, targeting the NF-kB and MK2 pathways greatly augment the suppressive effect of MAPK inhibitors. Notably, the mechanisms via which MK2 promotes PDAC cell survival are mediated through phospho-activation of Heat shock protein 27 (Hsp27) and Beclin1, a critical mediator of autophagy. Intriguingly, silencing of TNFR1 abrogated induction of proactive autophagy following MAPK inhibition, strongly suggesting autocrine signaling as the inciting event of autophagy. Mechanistically, we showed that TNF signaling upregulate unfolded protein response (UPR) in PDAC cells, which is required for autophagy induction. Targeting MK2 abrogated transcription of key UPR genes and blocks subsequent autophagy. The combination of MK2 inhibitor ATI-450 and ERK inhibitor ulixertinib was more effective in curbing the growth of PDAC patient-derived xenograft in vivo and prolonged the survival of autochthonous PDAC mice (KPC model). Overall, our study provided novel insights on the mechanisms that drive protective autophagy following MAPK pathway inhibition and a rationale and feasible therapeutic combination that can be tested in clinical trials for PDAC patients. Citation Format: Iftikhar Ali Khawar, Qing Wei, Timothy Hung-Po Chen, Lin Lin, Patrick M. Grierson, Kian-Huat Lim. TNF-MK2 signaling drives protective autophagy following MAPK pathway inhibition in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3870.

NF-κB transcription factors, driven by the IRAK/IKK cascade, confer treatment resistance in pancreatic ductal adenocarcinoma (PDAC), a cancer characterized by near-universal KRAS mutation. Through reverse-phase protein array and RNA sequencing we discovered that IRAK4 also contributes substantially to MAPK activation in KRAS-mutant PDAC. IRAK4 ablation completely blocked RAS-induced transformation of human and murine cells. Mechanistically, expression of mutant KRAS stimulated an inflammatory, autocrine IL-1β signaling loop that activated IRAK4 and the MAPK pathway. Downstream of IRAK4, we uncovered TPL2 (also known as MAP3K8 or COT) as the essential kinase that propels both MAPK and NF-κB cascades. Inhibition of TPL2 blocked both MAPK and NF-κB signaling, and suppressed KRAS-mutant cell growth. To counter chemotherapy-induced genotoxic stress, PDAC cells upregulated TLR9, which activated prosurvival IRAK4/TPL2 signaling. Accordingly, a TPL2 inhibitor synergized with chemotherapy to curb PDAC growth in vivo. Finally, from TCGA we characterized 2 MAP3K8 point mutations that hyperactivate MAPK and NF-κB cascades by impeding TPL2 protein degradation. Cancer cell lines naturally harboring these MAP3K8 mutations are strikingly sensitive to TPL2 inhibition, underscoring the need to identify these potentially targetable mutations in patients. Overall, our study establishes TPL2 as a promising therapeutic target in RAS- and MAP3K8-mutant cancers and strongly prompts development of TPL2 inhibitors for preclinical and clinical studies.

While most monogenic diseases are caused by loss or reduction of protein function, the need for technologies that can selectively increase levels of protein in native tissues remains. Here we demonstrate that antisense-mediated modulation of pre-mRNA splicing can increase endogenous expression of full-length protein by preventing naturally occurring non-productive alternative splicing and promoting generation of productive mRNA. Bioinformatics analysis of RNA sequencing data identifies non-productive splicing events in 7,757 protein-coding human genes, of which 1,246 are disease-associated. Antisense oligonucleotides targeting multiple types of non-productive splicing events lead to increases in productive mRNA and protein in a dose-dependent manner in vitro. Moreover, intracerebroventricular injection of two antisense oligonucleotides in wild-type mice leads to a dose-dependent increase in productive mRNA and protein in the brain. The targeting of natural non-productive alternative splicing to upregulate expression from wild-type or hypomorphic alleles provides a unique approach to treating genetic diseases., Restoration of normal gene expression is one way to treat monogenic disorders. Here the authors target naturally occurring non-productive alternative splicing using antisense oligonucleotides to promote the production of functional proteins.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with limited therapeutic options. We evaluated the safety and efficacy of the aminopeptidase inhibitor tosedostat with capecitabine in advanced PDAC. Methods: We conducted a phase Ib/II trial of tosedostat with capecitabine as second-line therapy for advanced PDAC. Planned enrollment was 36 patients. Eligible patients were treated with capecitabine 1,000 mg/m 2 oral twice-daily days 1–14 and oral tosedostat in a dose de-escalation design on days 1–21 of each 21-day cycle. Primary endpoints were the recommended phase 2 dose (RP2D) and progression-free survival (PFS). Results: Sixteen patients were enrolled. Tosedostat 120 mg oral twice daily with capecitabine 1,000 mg/ 2 oral twice daily was the RP2D. There was one dose-limiting toxicity (DLT) (grade 3 acute coronary syndrome) during phase Ib. The most common treatment-related adverse events were gastrointestinal (nausea, diarrhea), cardiac [QTc prolongation, decreased ejection fraction (EF)], and fatigue. The median PFS was 7.1 months, and the median treatment failure free survival was 3 months. Eight patients experienced stable disease for greater than 3 months. The study was closed early due to lack of drug availability. Conclusions: Tosedostat with capecitabine displayed tolerable toxicity, and prolonged disease control in a subset of patients. These data encourage further exploration of aminopeptidase inhibitors in pancreatic cancer.

BACKGROUND AND AIMS: Checkpoint immunotherapy is largely ineffective in pancreatic ductal adenocarcinoma (PDAC). The innate immune NF-κB pathway promotes PDAC cell survival, stromal fibrosis and is driven by IRAK4, but its impact on tumor immunity has not been directly investigated. METHODS: We interrogated the TCGA data to identify the correlation between NF-κB and T cell signature, and a PDAC tissue microarray (TMA) to correlate IRAK4 activity with CD8(+) T cells abundance. We performed RNAseq on IRAK4-deleted PDAC cells, and single cell RNAseq on autochthonous KPC (p48-Cre/TP53(f/f)/LSL-KRAS(G12D)) mice treated with an IRAK4 inhibitor. We generated conditional IRAK4-deleted KPC mice and complementarily used IRAK4 inhibitors to determine the impact of IRAK4 on T cell immunity. RESULTS: We found positive correlation between NF-κB activity, IRAK4 and T cell exhaustion from TCGA. We observed inverse correlation between phosphorylated IRAK4 and CD8(+) T cell abundance in a PDAC TMA. Loss of IRAK4 abrogates NF-κB activity, several immunosuppressive factors, checkpoint ligands and hyaluronan synthase 2 (HAS2), all of which drive T cell dysfunction. Accordingly, conditional deletion or pharmacologic inhibition of IRAK4 markedly decreased tumor desmoplasia and increased the abundance and activity of infiltrative CD4(+) and CD8(+) T cells in KPC tumors. Single cell RNAseq showed myeloid and fibroblast reprograming towards acute inflammatory responses following IRAK4 inhibition. These changes set the stage for successful combination of IRAK4 inhibitors with checkpoint immunotherapy, resulting in excellent tumor control and markedly prolonged survival of KPC mice. CONCLUSION: IRAK4 drives T cell dysfunction in PDAC and is a novel, promising immunotherapeutic target.

Ablative radiation therapy for borderline resectable or locally advanced pancreatic ductal adenocarcinoma (BR/LA-PDAC) may limit concurrent chemotherapy dosing and usually is only safely deliverable to tumors distant from gastrointestinal organs. Magnetic resonance guided radiation therapy may safely permit radiation and chemotherapy dose escalation.We conducted a single-arm phase I study to determine the maximum tolerated dose of ablative hypofractionated radiation with full-dose gemcitabine/nab-paclitaxel in patients with BR/LA-PDAC. Patients were treated with gemcitabine/nab-paclitaxel (1000/125 mg/mThirty patients enrolled (March 2015-February 2019), with 26 evaluable patients (2 progressed before radiation, 1 was determined ineligible for radiation during planning, 1 withdrew consent). One DLT was observed. The DLT rate was 14.1% (3.3%-24.9%) with a maximum tolerated dose of gemcitabine/nab-paclitaxel (1000/100 mg/mFull-dose gemcitabine/nab-paclitaxel with ablative magnetic resonance guided radiation therapy dosing is safe in patients with BR/LA-PDAC, with promising LPFS and DMFS.

Gallbladder cancer (GBC) is a biological, anatomical, and clinically distinct subset of biliary tract cancers (BTC), which also include extra- and intra-hepatic cholangiocarcinoma. The advent of next-generation sequencing (NGS) clearly shows that GBC is genetically different from cholangiocarcinoma. Although GBC is a relatively rare cancer, it is highly aggressive and carries a grave prognosis. To date, complete surgical resection remains the only path for cure but is limited to patients with early-stage disease. The majority of the patients are diagnosed at an advanced, inoperable stage when systemic treatment is administered as an attempt to enable surgery or for palliation. Gemcitabine and platinum-based chemotherapies have been the main treatment modality for unresectable, locally advanced, and metastatic gallbladder cancer. However, over the past decade, the treatment paradigm has evolved. These include the introduction of newer chemotherapeutic strategies after progression on frontline chemotherapy, incorporation of targeted therapeutics towards driver mutations of genes including HER2, FGFR, BRAF, as well as approaches to unleash host anti-tumor immunity using immune checkpoint inhibitors. Notably, due to the rarity of BTC in general, most clinical trials included both GBC and cholangiocarcinomas. Here, we provide a review on the pathogenesis of GBC, past and current systemic treatment options focusing specifically on GBC, clinical trials tailored towards its genetic mutations, and emerging treatment strategies based on promising recent clinical studies.

111 Background: Navicixizumab is an anti-delta-like ligand 4 (DLL4)/vascular endothelial growth factor bispecific antibody which has shown promising preclinical and clinical activity in multiple tumor types. The blockade of DLL4, a transmembrane protein acting as a ligand for Notch receptors 1 and 4, causes tumor differentiation and reduction in cancer stem cells, as well as repolarization of the tumor microenvironment. Methods: We conducted a phase 1b single-arm dose escalation study combining navicixizumab with FOLFIRI or FOLFOX as second line treatment for patients with metastatic colorectal cancer (CRC). Patients were dosed either at 3 or 4 mg/kg intravenously once every 2 weeks (Q2W). The primary endpoint was the maximum tolerated dose of navicixizumab in combination with FOLFOX or FOLFIRI. Results: Between December 2016 and August 2018, 15 patients received navicixizumab plus FOLFIRI. Median patient age was 55 years, and nine of 15 patients were female (60%). Three patients were enrolled in the 3 mg/kg dose-escalation cohort, 2 patients were enrolled in the 4-mg/kg dose-escalation cohort, and 10 patients were enrolled in the 3 mg/kg dose expansion cohort. No DLTs were observed in this study. The median duration of navicixizumab therapy was 127 days. Hypertension was the most frequent all-grade treatment-related adverse event reported (12 of 15 patients, 80%), followed by fatigue (8 of 15 patients, 53.3%). Grade 3 hypertension was reported in 3 patients (20%). Eight of the 15 patients (53.3%) experienced at least one serious adverse event, resulting in discontinuation of study for 6 (40%) patients. One patient had a bowel perforation. One patient (3 mg/kg cohort) experienced a treatment-related grade 5 cardiopulmonary arrest resulting in death. The best overall response was partial response in three patients (20%). Eleven patients (73.3%) had stable disease and one (6.7%) experienced disease progression. Conclusions: This study was stopped early to focus on the development of navicixizumab in ovarian cancer. Although the disease control rate was promising, further evaluation is needed to determine whether navicixizumab delivers efficacy greater than chemotherapy alone and assess tolerability of this regimen. [Table: see text]

Combination chemotherapies remain the cornerstone treatment for pancreatic ductal adenocarcinoma (PDAC), but de novo and acquired resistance is near universal. In this study we aimed to identify and characterize resistance mechanisms to FIRINOX chemotherapy regimen (a combination of 5-fluorouracil, irinotecan and oxaliplatin), because it is the most aggressive regimen currently used clinically for patients with PDAC. Using an unbiased reverse-phase protein array, we detected phospho-activation of Heat shock protein 27 (Hsp27) as the most upregulated event following FIRINOX treatment in PDAC cells. Silencing HSP27 by RNA interference or by small molecule inhibitor enhanced apoptosis in vitro caused by FIRINOX. Mechanistically, FIRINOX upregulated tumor necrosis factor alpha (TNFα), causing autocrine phosphorylation and activation of transforming growth factor-β-activated kinase 1 (TAK1), MAPK Activated Protein Kinase 2 (MAPKAPK2 or MK2) and ultimately Hsp27. Targeting MK2, the kinase that directly phosphorylates Hsp27, abrogated Hsp27 activation, sensitized PDAC cells to apoptosis, and suppressed SN-38-induced protective autophagy in vitro, in part by blocking phospho-activation of Beclin1. In an autochthonous PDAC mouse model, the MK2 inhibitor ATI-450 decreased PDAC development and progression. When combined with FIRINOX, ATI-450 eliminated most PDAC foci and marked prolonged mouse survival without causing additional toxicity. Last, we found that high phospho-MK2 expression in tumors was associated with inferior survival of patients with PDAC. Our study identified MK2 as a mediator of genotoxic stress-induced activation of pro-survival pathways and provides preclinical support for combining an MK2 inhibitor with FIRINOX-based chemotherapies to treat PDAC.

Tumor-associated macrophages (TAMs) are involved in many aspects of cancer progression and correlate with poor clinical outcomes in many cancer types, including pancreatic ductal adenocarcinomas (PDACs). Previous studies have shown that TAMs can populate PDAC tumors not only by monocyte recruitment but also by local proliferation. However, the impact local proliferation might have on macrophage phenotype and cancer progression is unknown. Here, we utilized genetically engineered cancer models, single-cell RNA-sequencing data, andin vitrosystems to show that proliferation of TAMs was driven by colony stimulating factor-1 (CSF1) produced by cancer-associated fibroblasts. CSF1 induced high levels of p21 in macrophages, which regulated both TAM proliferation and phenotype. TAMs in human and mouse PDACs with high levels of p21 had more inflammatory and immunosuppressive phenotypes. The p21 expression in TAMs was induced by both stromal interaction and/or chemotherapy treatment. Finally, by modeling p21 expression levels in TAMs, we found that p21-driven macrophage immunosuppressionin vivodrove tumor progression. Serendipitously, the same p21-driven pathways that drive tumor progression, also drive response to CD40 agonist. These data suggest that stromal or therapy-induced regulation of cell cycle machinery can regulate both macrophage-mediated immune suppression and susceptibility to innate immunotherapy.SummaryTAMs are indicative of poor clinical outcomes and in PDAC their number is sustained in part by local proliferation. This study shows that stromal desmoplasia drives local proliferation of TAMs, and induces their immunosuppressive ability through altering cell cycle machinery, including p21 expression. Serendipitously, these changes in p21 in TAMs also potentially render tumors more sensitive to CD40 agonist therapy.

Simple Summary Defining the mechanisms underlying the heterogeneous cancer cell response to DNA damage is important for our ability to tailor individualized strategies to improve chemotherapy efficacy. In this study, we focus on a central DNA repair regulator named VCP whose loss-of-function has been shown by prior studies to increase chemotherapy effects. However, since VCP participates in numerous cellular processes, its total expression level cannot differentiate between VCP-dependent vs. -independent tumors with specific regard to chemotherapy-induced DNA repair. Here, by examining cell lines and patient samples of pancreatic ductal adenocarcinoma (PDAC), we made two major observations. First, DNA damage-induced Ser784 phosphorylation is essential for DNA repair by VCP and drives chemo-resistance. Second, pSer784-VCP instead of total VCP protein predicts cellular reliance on VCP function during DNA repair and consequently chemo-sensitizing effects of VCP inhibition. Thus, pSer784-VCP may be a potential predictive biomarker and sensitizing target for cancer chemotherapy treatments. Abstract Pancreatic ductal adenocarcinoma (PDAC) patients have a dismal prognosis due in large part to chemotherapy resistance. However, a small subset containing defects in the DNA damage response (DDR) pathways are chemotherapy-sensitive. Identifying intrinsic and therapeutically inducible DDR defects can improve precision and efficacy of chemotherapies for PDAC. DNA repair requires dynamic reorganization of chromatin-associated proteins, which is orchestrated by the AAA+ ATPase VCP. We recently discovered that the DDR function of VCP is selectively activated by Ser784 phosphorylation. In this paper, we show that pSer784-VCP but not total VCP levels in primary PDAC tumors negatively correlate with patient survival. In PDAC cell lines, different pSer784-VCP levels are induced by genotoxic chemotherapy agents and positively correlate with genome stability and cell survival. Causal effects of pSer784-VCP on DNA repair and cell survival were confirmed using VCP knockdown and functional rescue. Importantly, DNA damage-induced pSer784-VCP rather than total VCP levels in PDAC cell lines predict their chemotherapy response and chemo-sensitizing ability of selective VCP inhibitor NMS-873. Therefore, pSer784-VCP drives genotoxic chemotherapy resistance of PDAC, and can potentially be used as a predictive biomarker as well as a sensitizing target to enhance the chemotherapy response of PDAC.

Folate receptor α (FRα) is a well-studied tumor biomarker highly expressed in many epithelial tumors such as breast, ovarian, and lung cancers. Mirvetuximab soravtansine (IMGN853) is the antibody-drug conjugate of FRα-binding humanized monoclonal antibody M9346A and cytotoxic maytansinoid drug DM4. IMGN853 is currently being evaluated in multiple clinical trials, in which the immunohistochemical evaluation of an archival tumor or biopsy specimen is used for patient screening. However, limited tissue collection may lead to inaccurate diagnosis due to tumor heterogeneity. Herein, we developed a zirconium-89 (89Zr)-radiolabeled M9346A (89Zr-M9346A) as an immuno-positron emission tomography (immuno-PET) radiotracer to evaluate FRα expression in triple-negative breast cancer (TNBC) patients, providing a novel means to guide intervention with therapeutic IMGN853. In this study, we verified the binding specificity and immunoreactivity of 89Zr-M9346A by in vitro studies in FRαhigh cells (HeLa) and FRαlow cells (OVCAR-3). In vivo PET/computed tomography (PET/CT) imaging in HeLa xenografts and TNBC patient-derived xenograft (PDX) mouse models with various levels of FRα expression demonstrated its targeting specificity and sensitivity. Following PET imaging, the treatment efficiencies of IMGN853, pemetrexed, IMGN853 + pemetrexed, paclitaxel, and saline were assessed in FRαhigh and FRαlow TNBC PDX models. The correlation between 89Zr-M9346A tumor uptake and treatment response using IMGN853 in FRαhigh TNBC PDX model suggested the potential of 89Zr-M9346A PET as a noninvasive tool to prescreen patients based on the in vivo PET imaging for IMGN853-targeted treatment.

Simple Summary Liver metastasis is a major therapeutic challenge and common cause of death for patients with colorectal cancer. While systemic treatment especially chemotherapy remains the mainstay of treatment, selected patients with liver-only metastasis may further benefit from liver-directed therapies. Direct infusion of chemotherapy into the liver metastases via an implantable hepatic arterial infusion pump (HAIP) is potentially an effective way to improve treatment response and survival in selected patients. Here, we reviewed the literature utilizing HAIP as a liver-directed modality alone and in combination with systemic chemotherapy. We discussed two cases who were successfully treated with this combinatorial approach and achieved remission or prolongation of disease control. We discussed the limitations, toxicities of combined systemic and HAIP modalities. Lastly, we provided insights on the use of HAIP in the modern era of systemic treatment for colorectal cancer patients with liver metastasis. Abstract Colorectal cancer (CRC) is the third most prevalent malignancy and the second most common cause of death in the US. Liver is the most common site of colorectal metastases. About 13% of patients with colorectal cancer have liver metastasis on initial presentation and 50% develop them during the disease course. Although systemic chemotherapy and immunotherapy are the mainstay treatment for patients with metastatic disease, for selected patients with predominant liver metastasis, liver-directed approaches may provide prolonged disease control when combined with systemic treatments. Hepatic artery infusion pump (HAIP) chemotherapy is an approach which allows direct infusion of chemotherapeutic into the liver and is especially useful in the setting of multifocal liver metastases. When combined with systemic chemotherapy, HAIP improves the response rate, provides more durable disease control, and in some patients leads to successful resection. To ensure safety, use of HAIP requires multidisciplinary collaboration between interventional radiologists, medical oncologists, hepatobiliary surgeons and treatment nurses. Here, we review the benefits and potential risks with this approach and provide our single institution experience on two CRC patients successfully treated with HAIP in combination with systemic chemotherapy. We provide our recommendations in adopting this technique in the current era for patient with colorectal liver metastases.