KRAS-mutant Pancreatic Cancer Research Articles

Targeting p130Cas- and microtubule-dependent MYC regulation sensitizes pancreatic cancer to ERK MAPK inhibition.

SUMMARYTo identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determine that SRC-inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-β-catenin-dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the βIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function decreases levels of MYC protein in a calpain-dependent manner and potently sensitizes pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperates to reduce MYC protein and synergistically suppress the growth of KRAS mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation.

Eicosapentaenoic Acid Inhibits KRAS Mutant Pancreatic Cancer Cell Growth by Suppressing Hepassocin Expression and STAT3 Phosphorylation.

Background: The oncogenic Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation was reported to be the signature genetic event in most cases of pancreatic ductal adenocarcinoma (PDAC). Hepassocin (HPS/FGL1) is involved in regulating lipid metabolism and the progression of several cancer types; however, the underlying mechanism of HPS/FGL1 in the KRAS mutant PDAC cells undergoing eicosapentaenoic acid (EPA) treatment remains unclear. Methods: We measured HPS/FGL1 protein expressions in a human pancreatic ductal epithelial (HPNE) normal pancreas cell line, a KRAS-wild-type PDAC cell line (BxPC-3), and KRAS-mutant PDAC cell lines (PANC-1, MIA PaCa-2, and SUIT-2) by Western blot methods. HEK293T cells were transiently transfected with corresponding KRAS-expressing plasmids to examine the level of HPS expression with KRAS activation. We knocked-down HPS/FGL1 using lentiviral vectors in SUIT-2 cells and measured the cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenicity assays. Furthermore, a lipidomic analysis was performed to profile changes in lipid metabolism after HPS/FGL1 knockdown. Results: We found that the HPS/FGL1 level was significantly upregulated in KRAS-mutated PDAC cells and was involved in KRAS/phosphorylated (p)-signal transduction and activator of transcription 3 (STAT3) signaling, and the knockdown of HPS/FGL1 in SUIT-2 cells decreased cell proliferation through increasing G2/M cell cycle arrest and cyclin B1 expression. In addition, the knockdown of HPS/FGL1 in SUIT-2 cells significantly increased omega-3 polyunsaturated fatty acids (PUFAs) and EPA production but not docosahexaenoic acid (DHA). Moreover, EPA treatment in SUIT-2 cells reduced the expression of de novo lipogenic protein, acetyl coenzyme A carboxylase (ACC)-1, and decreased p-STAT3 and HPS/FGL1 expressions, resulting in the suppression of cell viability. Conclusions: Results of this study indicate that HPS is highly expressed by KRAS-mutated PDAC cells, and HPS/FGL1 plays a crucial role in altering lipid metabolism and increasing cell growth in pancreatic cancer. EPA supplements could potentially inhibit or reduce ACC-1-involved lipogenesis and HPS/FGL1-mediated cell survival in KRAS-mutated pancreatic cancer cells.

Nrf2 Activation Sensitizes K-Ras Mutant Pancreatic Cancer Cells to Glutaminase Inhibition.

Pancreatic cancer remains intractable owing to the lack of effective therapy for unresectable cases. Activating mutations of K-ras are frequently found in pancreatic cancers, but these have not yet been targeted by cancer therapies. The Keap1-Nrf2 system plays a crucial role in mediating the oxidative stress response, which also contributes to cancer progression. Nrf2 activation reprograms the metabolic profile to promote the proliferation of cancer cells. A recent report suggested that K-ras- and Nrf2-active lung cancer cells are sensitive to glutamine depletion. This finding led to the recognition of glutaminase inhibitors as novel anticancer agents. In the current study, we used murine pancreatic cancer tissues driven by mutant K-ras and p53 to establish cell lines expressing constitutively activated Nrf2. Genetic or pharmacological Nrf2 activation in cells via Keap1 deletion or Nrf2 activation sensitized cells to glutaminase inhibition. This phenomenon was confirmed to be dependent on K-ras activation in human pancreatic cancer cell lines harboring mutant K-ras, i.e., Panc-1 and MiaPaCa-2 in response to DEM pretreatment. This phenomenon was not observed in BxPC3 cells harboring wildtype K-ras. These results indicate the possibility of employing Nrf2 activation and glutaminase inhibition as novel therapeutic interventions for K-ras mutant pancreatic cancers.

Fats and Mets, KRAS-Driven Lipid Dysregulation Affects Metastatic Potential in Pancreatic Cancer.

In this issue of Cancer Research, Rozeveld and colleagues present intriguing evidence of the importance of lipid droplets and hormone-sensitive lipase (HSL) in regulating the aggressive nature of pancreatic cancer. Initially demonstrating a dependency of preloaded lipids on an invasive phenotype, the authors then establish that oncogenic KRAS mutation downregulates HSL, thereby facilitating lipid storage during steady state. Thereafter, a phenotypic switch to oxidative metabolism with lipid utilization to fuel invasion and metastasis occurs. Experimentally, blocking the KRAS-HSL axis results in fewer lipid droplets, as well as metabolic reprogramming of the invasive cell phenotype, effectively reducing invasive capacity of KRAS-mutant pancreatic cancer. Of note, HSL overexpression in tumor cells also inhibited invasion, due to depletion of lipid droplets and the stored lipids, which are essential during invasion. Collectively, these novel findings highlight the importance of energy metabolism and its dynamic regulation in the evolution of the metastatic capacity of pancreatic cancer.See related article by Rozeveld et al., p. 4932.

Abstract PO-032: Targeting the KRAS dimerization interface enhances T-cell mediated anti-tumor response in vivo

Abstract RAS is the most frequently mutated human oncogene and about 20% of all human cancers harbor mutations in one of three RAS oncogenes (K, N and H-RAS) with pancreatic cancers harboring RAS mutations in >90% of tumors. Therapeutically targeting RAS has been highly challenging and other than allele specific KRAS (G12C) inhibitors, no direct RAS inhibitor has been approved for clinical trial. However, KRAS (G12C) mutations are less abundant in some cancers like pancreatic cancer, thus there remains an unmet need for inhibitors that target the more common RAS mutants. This demand devising novel strategies to identify new approaches to inhibit RAS. Previously, we developed the H/KRAS-specific monobody called NS1 that allosterically inhibit RAS-mediated signaling by targeting the α4-α5 dimerization interface. When used as a genetically encoded intracellular reagent, NS1 inhibited the growth of H/KRAS-mutant human tumor lines both in vitro and in xenograft tumor models. However, these tumor models lack a functional immune system. Here, we evaluated the potency of targeting KRAS dimerization interface with NS1 in an immune-competent murine model of pancreatic cancer. Although human and murine KRAS differ by a single amino acid [Asp (D) to Glu (E)] at position 132 in the NS1 binding region of the allosteric lobe, NS1 bound similarly to human and murine KRAS both in vitro and in vivo. Furthermore, NS1 expression inhibited ERK-MAPK activation, proliferation and anchorage-independent growth of engineered KPC cells derived from pancreatic tumor in the KPC genetic mouse model. Finally, when these engineered KPC cells were injected orthotopically in immune-competent C57/B6 mice, we observed a dramatic decrease in tumor growth kinetics and tumor burden in NS1 expressing cohorts. Similarly, NS1 expression resulted in decreased ERK-MAPK activation and increased apoptosis. NS1 expressing tumors were characterized by increased infiltration of helper CD4+ T cells and macrophage. Thus, inhibition of mutant KRAS by NS1 results in an enhanced anti-tumor immune response in a syngeneic model for pancreatic cancer. These results establish that targeting α4-α5 dimerization interface of RAS can be a viable therapeutic option for targeting KRAS-mutant pancreatic cancer. Citation Format: Imran Khan, Julia Lefler, Catherine Marelia, Mariyam Zuberi, Eric Denbaum, Akiko Koide, Cynthia Timmers, Michael Ostrowski, Shohei Koide, John O’Bryan. Targeting the KRAS dimerization interface enhances T-cell mediated anti-tumor response in vivo [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-032.

Abstract A02: SYK kinase inhibition causes autophagy pathway activation via suppression of mTORC1 in KRAS-mutant pancreatic cancer cells

Abstract The spleen tyrosine kinase (SYK) is highly expressed in a subset of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines. This prompted our interest in investigating SYK as a candidate therapeutic target in PDAC cell lines. Here, we use a selective SYK kinase inhibitor (PRT-062607) in pharmacologic and mechanistic studies of SYK kinase signaling in PDAC cell lines. SYK kinase inhibition causes dose-dependent decreases in cell proliferation of “SYK-high” PDAC cell lines YAPC and DAN-G. Furthermore, combined SYK and MEK inhibition causes additive effects on reducing cell proliferation. Mechanistically, SYK inhibition causes reduced S6K and rpS6 phosphorylation, which is independent of MEK/ERK and PI3K/AKT signaling. Therefore, SYK activates mTORC1 activity through an unknown mechanism. The mTORC1 complex is a key activator of the macroautophagy pathway (autophagy), which is implicated as a driver of PDAC progression. However, autophagy can cause tumor suppression in some contexts. We demonstrate that SYK inhibition or shRNA-mediated depletion results in increased autolysosomal biogenesis and autophagic flux that occur with latency following treatment of cells with the SYK inhibitor. These effects are partly mediated by the nuclear localization and activation of MITF, which is a key transcriptional regulator of genes that control lysosomal biogenesis and autophagy. Taken together, our results demonstrate that SYK is a positive regulator of mTORC1 activity in a subset of PDAC cell lines. SYK kinase inhibition or shRNA-mediated depletion causes a strong reduction in mTORC1 activity and subsequent MITF activation and nuclear localization. The net effect is increased autolysosomal biogenesis and autophagic flux in YAPC and DAN-G PDAC cell lines. Therefore, SYK inhibition in combination with KRAS pathway inhibitors may serve as a potential therapeutic strategy to perturb autophagy pathway homeostasis and pancreatic cancer cell survival. Citation Format: Kevin L. Hua, Michelle Pan, Minoo Rafati, Anurag Singh. SYK kinase inhibition causes autophagy pathway activation via suppression of mTORC1 in KRAS-mutant pancreatic cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A02.

Effects of Co-occurring Genomic Alterations on Survival in Patients with KRAS-Mutant Pancreatic Cancer

We report the case of a young patient with left breast cancer with stage IIB (T2N1M0), negative hormone receptors, HER2-positive and no particular medical history. The patient was particularly sensitive to the neurological and allergic-type toxicity of the adjuvant treatment she received and it required the stopping and successive change of the various prescribed protocols.

Controlled coupling of an ultrapotent auristatin warhead to cetuximab yields a next-generation antibody-drug conjugate for EGFR-targeted therapy of KRAS mutant pancreatic cancer

BackgroundAntibody-drug conjugate (ADC) construction poses numerous challenges that limit clinical progress. In particular, common bioconjugation methods afford minimal control over the site of drug coupling to antibodies. Here, such difficulties are overcome through re-bridging of the inter-chain disulfides of cetuximab (CTX) with auristatin-bearing pyridazinediones, to yield a highly refined anti-epidermal growth factor receptor (EGFR) ADC.MethodsIn vitro and in vivo assessment of ADC activity was performed in KRAS mutant pancreatic cancer (PaCa) models with known resistance to CTX therapy. Computational modelling was employed for quantitative prediction of tumour response to various ADC dosing regimens.ResultsSite-selective coupling of an auristatin to CTX yielded an ADC with an average drug:antibody ratio (DAR) of 3.9, which elicited concentration- and EGFR-dependent cytotoxicity at sub-nanomolar potency in vitro. In human xenografts, the ADC inhibited tumour growth and prolonged survival, with no overt signs of toxicity. Key insights into factors governing ADC efficacy were obtained through a robust mathematical framework, including target-mediated dispositional effects relating to antigen density on tumour cells.ConclusionsTogether, our findings offer renewed hope for CTX in PaCa therapy, demonstrating that it may be reformatted as a next-generation ADC and combined with a predictive modelling tool to guide successful translation.

Effects of Co-occurring Genomic Alterations on Survival in Patients with KRAS-Mutant Pancreatic Cancer

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Targeting P130Cas- and Microtubule-Dependent MYC Regulation Sensitizes Pancreatic Cancer to ERK MAPK Inhibition

To identify therapeutic targets for KRAS-mutant pancreatic cancer, we conducted a druggable genome siRNA screen and determined that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identified BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determined that SRC inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-beta-catenin dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the betaIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function, decreased levels of MYC protein and potently sensitized pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperated to reduce MYC protein and to synergistically suppress the growth of KRAS-mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation.

Abstract 6216: Therapeutic potential of IAP inhibitor APG-1387 in combination with PARP- or MEK-targeted therapy, or chemotherapy in pancreatic cancer

Abstract APG-1387 is a small molecule antagonist of the inhibitor of apoptosis proteins (IAPs) which blocks the activity of IAPs family proteins (XIAP, cIAP-1, cIAP-2, and ML-IAP) and consequently induces apoptosis. It is currently in phase I clinical development in patients with solid tumors, including pancreatic cancer. Targeting IAPs has been reported to effectively inhibit tumor growth in the preclinical models of pancreatic cancer. Currently, PARP and MEK inhibitors are also in clinical development for pancreatic cancer with BRCA1/2 or KRAS mutations. To further explore the therapeutic potential of APG-1387 in combination with PARP or MEK inhibitors, a genomic biomarker guided avatar mouse trial (n=2) using pancreatic cancer patient-derived xenografts (PDX) was conducted to evaluate antitumor activity of APG-1387 in combination with (i) the PARP inhibitor, olaparib, in PDXs carrying BRCA1/2 mutations, and (ii) the MEK inhibitor, trametinib, in PDXs carrying KRAS mutations. The results revealed that APG-1387 improved the antitumor activity of olaparib in 4 out of 6 (67%) BRCA1/2 mutant PDXs with a 44% reduction of T/C (%) value (T, treatment group, C, vehicle control), including one partial tumor regression (PR). In combination with trametinib, the antitumor activity was improved in 4 out of 9 (44%) KRAS mutant PDXs, showing a 34% reduction in the T/C value. Furthermore, synergy of the combinations of APG-1387 with PARP or MEK inhibitor was confirmed in a follow-up efficacy study (n=5) using a PDX model carrying both BRCA2 and KRAS mutations. The molecular mechanisms underlying the synergy are currently being investigated. In summary, our results suggest that APG-1387 is a potential agent for pancreatic cancer treatment, ascribing to its potential synergistic antitumor effect by combining with either PARP inhibitors in BRCA1/2 mutant or MEK inhibitors in KRAS mutant pancreatic cancers. Overall, our data provide the scientific rationale for the future clinical development of these combinations in pancreatic cancer patients with distinct genomic alterations. Citation Format: Qiang Li, Douglas D. Fang, Shoulai Gu, Dajun Yang, Yifan Zhai. Therapeutic potential of IAP inhibitor APG-1387 in combination with PARP- or MEK-targeted therapy, or chemotherapy in pancreatic cancer [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 6216.

Abstract 987: Nrf2-dependent HO-1 positive macrophages accumulate in murine models of KRAS-mutated pancreatic cancer and promote tumorigenesis

Abstract The Nrf2 (NF-E2-related factor-2) transcription factor regulates oxidative/xenobiotic stress responses and can drive cancer progression, metastasis and chemoresistance. Nrf2 also attenuates inflammation through elimination of reactive oxygen species (ROS) and regulation of macrophage specific genes involved in oxidative/stress responses. The tumor microenvironment in pancreatic cancer is rich in macrophages that promote cancer progression and chemoresistance. Recent studies suggest that HO-1 positive macrophages are immunosuppressive, however the role of these macrophages in tumor progression and metastasis is not known. We hypothesized that the activation of Nrf2-dependent macrophages expressing HO-1 in the pancreas is essential for tumor development and metastasis.Heme-oxygenase 1 (HO-1) is an inducible enzyme, classical expressed by anti-inflammatory macrophages, to degrade heme. HO-1 expression is dependent on the translocation of NRF2 to the nucleus and activation of redox-electrophilic stress genes. In RAW 267.4 cells or bone marrow derived macrophages stimulated with conditioned media from murine pancreatic cancer cells, HO-1 protein levels increased. In contrast, HO-1 expression in macrophages was not affected when these cells were treated with classical macrophage activators such as IL-6 and IL-4. Increased HO-1 protein expression was associated with higher NRF2 expression in the nuclear fraction.Pancreatitis, or inflammation of the pancreas, is a known risk factor for pancreatic cancer, as hereditary pancreatitis increases the lifetime risk of developing cancer by 40-50%. In mice, chronic or acute pancreatitis promotes mutant Kras-mediated development of pancreatic cancer. LSL-KrasG12D/+;Pdx-1-Cre (KC) and Nrf2 whole body knockout (KO) mice were stimulated with cerulean (75 ug/kg injected intra peritoneally, every hour for 8 hours for 2 consecutive days) to induce pancreatitis. Pancreatitis, human and murine, is associated with an increased infiltration of macrophages to the pancreas during the acute phase of the disease. After 72 hours post-cerulean stimulation, macrophage infiltration into the pancreas was analyzed by flow cytometry. KC mice showed a significantly (P<0.05) higher infiltration of macrophages when compared with Nrf2 KO or wild type mice (51.1% vs 10.7% vs 21.5% of all immune cells in the pancreas). Additionally, pancreas of KC mice expressed higher levels of HO-1 protein when compared with NRF2 KO and wild-type mice. KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) mice will succumb to pancreatic cancer at an average of 23 weeks of age. At 12 weeks of age, the levels of HO-1 in the pancreas are increased, promoting tumorigenesis. Additionally, the levels of HO-1 in the liver, one of the primary sites for metastasis, are increased at 20 weeks, surrounding early lesions. These studies suggest that Nrf2 activation in macrophages, which elevates HO-1 expression, is hijacked by Kras-mutated epithelial cells in order to promote tumorigenesis and metastasis. Citation Format: Ana S. Mendes Leal, Karen T. Liby. Nrf2-dependent HO-1 positive macrophages accumulate in murine models of KRAS-mutated pancreatic cancer and promote tumorigenesis [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 987.

Nano-delivery of Gemcitabine Derivative as a Therapeutic Strategy in a Desmoplastic KRAS Mutant Pancreatic Cancer.

Pancreatic ductal adenocarcinoma remains one of the challenging malignancies to treat, and chemotherapy is the primary treatment strategy available to most patients. Gemcitabine, one of the oldest chemotherapeutic drugs approved for pancreatic cancer, has limited efficacy, due to low drug distribution to the tumor and chemoresistance following therapy. In this study, we delivered gemcitabine monophosphate using lipid calcium phosphate nanoparticles, to desmoplastic pancreatic tumors. Monophosphorylation is a critical, rate-limiting step following cellular uptake of gemcitabine and precursor of the pharmacologically active gemcitabine triphosphate. Our drug delivery strategy enabled us to achieve robust tumor regression with a low parenteral dose in a clinically relevant, KRAS mutant, syngeneic orthotopic allograft, lentivirus-transfected KPC cell line-derived model of pancreatic cancer. Treatment with gemcitabine monophosphate significantly increased apoptosis of cancer cells, enabled reduction in the proportion of immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells, and did not increase expression of cancer stem cell markers. Overall, we could trigger a strong antitumor response in a treatment refractory PDAC model, while bypassing critical hallmarks of gemcitabine chemoresistance.

Pharmacodynamic effects in blood and tumor tissue of eftozanermin alfa, a tumor necrosis factor-related apoptosis-inducing ligand receptor agonist.

e15668 Background: Eftozanermin alfa (eftoza; formerly known as ABBV-621), a 2nd-generation tumor necrosis factor-related apoptosis-inducing ligand receptor agonist, is being evaluated in previously treated solid and hematologic malignancies (NCT03082209). In a dose-expansion cohort, patients (pts) with KRAS-mutant colorectal cancer (n = 24) and pancreatic cancer (n = 24) were evaluated at 3 dose levels with 12 mandatory paired biopsies per tumor type (pretreatment [Tx] and on-Tx collection). Following eftoza dosing, RNA and protein expression including posttranslational modifications were assessed in tumor biopsies to understand the target engagement and downstream pathway activation. Plasma was evaluated for changes in somatic mutant allele frequency and M30, M65 (circulating apoptotic markers). Methods: Biopsies were collected anytime during the screening period (pre-Tx) and 24±4 h following 2nd or 3rd infusion (on-Tx). Of the requested 4–6 fresh biopsy cores, 1–2 cores were collected as formalin fixed paraffin embedded (FFPE) and the rest were frozen tissue. FFPE tissue was analyzed by multiplex immunohistochemistry (IHC) and RNAseq; reverse phase protein array was used for frozen cores. Plasma was collected at cycle 1 predose and 2, 8, 24, 48, and 168 h postdose and analyzed for M30, M65 (by ELISA) and circulating tumor DNA (64-gene PlasmaSELECT assay). Results: Twenty-five pts consented to biopsies; paired biopsies were obtained from 16 pts at a 64% success rate: FFPE (n = 15) and frozen cores (n = 12). Tumor cells were detected in 11/15 (73%) FFPE and 4/12 (33%) frozen cores. Increase in M30, activated caspases, and cleaved PARP levels was observed in on-Tx biopsy samples compared with pre-Tx, thus serving as evidence for apoptosis induction in tumors following eftoza dosing. Changes in the tumor microenvironment were observed post-Tx by RNAseq and multiplex IHC (eg, CD68 level). Downregulation of prosurvival signaling pathways (eg, AKT/MEK) was also observed following eftoza dosing. Thirteen out of 16 pts showed transient increase in mutant allele fractions post eftoza Tx that correlated with increased plasma circulating tumor markers M30 and M65 at similar time points, suggesting activation of apoptosis pathway. Increase in M30, M65 levels also preceded increase in liver enzymes (ALT/AST) at 2, 48 hr post-Tx. Conclusions: Pharmacodynamic effect of eftoza was successfully demonstrated in blood and tumor tissue, including induction of apoptosis and modulation of PI3K and MEK signaling pathways. Clinical trial information: NCT03082209.

Abstract IA25: Pancreatic cancer models and medicine

Abstract Since the most common KRAS oncoproteins lack therapeutic antagonists, we utilized mouse and organoid models to discover new characteristics of oncogenic KRAS that could represent new therapeutic opportunities in pancreatic ductal adenocarcinoma (PDAC). Continuous oncogenic Kras expression is required in PDAC mouse models to support the characteristic desmoplastic stroma that impairs drug delivery, suppresses the immune response, and promotes tumor progression. We identified the secretion of Tgf-β and IL-1α by neoplastic cells as ligands that produce stroma by stimulating the differentiation of pancreatic stellate cells into myofibroblasts (myCAFs) and inflammatory fibroblasts (iCAFs), respectively. Mutual antagonism between these two ligands led to the spatial patterning of these two fibroblast populations, with myCAFs juxtaposed to neoplastic cells while iCAFs located more distantly. Genetic and pharmacologic perturbation of iCAFs support the role of this subpopulation in PDAC progression, nominating them as a cellular target in Kras-dependent PDAC. Secondly, taking a cell-intrinsic approach, we found that oncogenic KRAS induced BNIP3L/Nix to stimulate selective mitophagy. Nix induction restricted flux of glucose to the mitochondria and enhanced cellular redox control, and loss of Nix reverted these effects and decreased cell proliferation in vitro. Furthermore, loss of Nix markedly delayed progression of pancreatic cancer in vivo. Our results reveal the Kras-Nix mitophagy program as a new mechanism to promote aerobic glycolysis, redox homeostasis, and disease progression in KRAS-mutant pancreatic cancer, and motivate the exploration of methods to antagonize Nix pharmacologically. Citation Format: David Tuveson. Pancreatic cancer models and medicine [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA25.

Abstract B31: Combination inhibitor strategies targeting KRAS effector signaling in KRAS-mutant pancreatic cancer

Abstract With KRAS mutations found in 95% of pancreatic ductal adenocarcinoma (PDAC), an effective anti-KRAS therapeutic strategy is anticipated to make a significant impact on the treatment of PDAC. Among the major directions currently being pursued, inhibitors of KRAS effector signaling are believed to be the most promising. However, as monotherapy, effector inhibitors have not demonstrated significant activity and concerns with normal tissue toxicity remain. Combination inhibitor strategies are considered the best approach to overcome these limitations. To identify effector inhibitor-based combinations, we applied a 525-oncology drug screen to identify combinations that enhanced the cytotoxic activity of inhibitors of the RAF-MEK-ERK and the PI3K-AKT-mTOR effector pathways. While many cytotoxic combinations were identified with RAF effector pathway inhibitors, few were identified with PI3K pathway inhibitors. The same classes of inhibitors were identified with RAF, MEK and ERK inhibitors. In addition to inhibitors of the PI3K-AKT-mTOR pathway, microtubule, HDAC and HSP90 inhibitors synergistically enhanced RAF and ERK inhibitor antitumor activity. Addressing a basis for synergy, we found that the different combinations showed enhanced loss of MYC protein, a key ERK substrate. In summary, our studies identify promising effector inhibitor-based combinations for PDAC treatment. Citation Format: Irem Ozkan-Dagliyan, Craig M. Goodwin, Kirsten L. Bryant, Samuel D. George, Kelly Lucas, Prson Gautam, Krister Wennerberg, Adrienne D. Cox, Channing J. Der. Combination inhibitor strategies targeting KRAS effector signaling in KRAS-mutant pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B31.

Abstract A44: SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS-mutant tumors treated with MEK inhibitors

Abstract FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTKs) are also feedback activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using a SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS mutant cancer cells and involves several RTKs including EGFR, FGFR, and MET. We further demonstrate this pathway feedback activation is mediated through mutant KRAS. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS mutant cancer cell proliferation in vitro and in vivo. These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic. Citation Format: Hengyu Lu, Chen Liu, Roberto Velazquez, Hongyun Wang, Lukas M. Dunkl, Malika Kazic-Legueux, Anne Haberkorn, Eric Billy, Eusebio Manchado, Saskia M. Brachmann, Susan Moody, Jeffrey A. Engelman, Peter S. Hammerman, Giordano Caponigro, Morvarid Mohseni, Huai-Xiang Hao. SHP2 inhibition overcomes RTK-mediated pathway reactivation in KRAS-mutant tumors treated with MEK inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A44.

Abstract IA13: Targeting redox dependencies in pancreatic cancer

Abstract Dysregulation of mRNA translation is a common feature of human cancers. As such, therapeutic agents that target components of the protein synthesis apparatus hold promise as anticancer drugs. We previously showed that protein synthesis is upregulated in Kras mutant pancreatic cancer cells in a manner that is redox-dependent (1). By modulating formation of the eIF4F complex, the PI3K/AKT and mTOR signaling pathways act as major regulators of global and mRNA-specific translation. Consistently, we observed that protein synthesis and the viability of pancreatic tumor organoids are selectively decreased by the pan-AKT inhibitor MK2206, an effect that was potentiated by co-treatment with the pro-oxidant BSO1. Treatment of KrasG12D;p53R172H;PdxCre (KPC) mice with this drug combination resulted in suppressed tumor kinetics and modest improvement in survival. Inhibition of AKT/mTOR blunts cap-dependent translation initiation but also perturbs a plethora of other cellular functions. We thus investigated more selective means of targeting the translation machinery that do not entail wholesale ablation of the AKT/mTOR pathway. The RNA helicase eIF4A initiates translation by unwinding highly structured 5′-untranslated regions (UTRs) in mRNAs. Using polysome profiling followed by deep sequencing, we found that the translation of eIF4A-sensitive mRNAs was selectively increased in Kras-mutant tumor versus normal organoids. The rocaglate, CR-13-1b, is a compound that selectively inhibits the eIF4A helicase and displayed high toxicity in Kras mutant tumor organoids, but not in normal organoids. Polysome sequencing revealed that CR131b selectively modulates translational efficiencies in tumor but not normal organoids. Interestingly, this included multiple mRNAs encoding proteins involved in mitochondrial activity and redox homeostasis. As a single agent, CR131b treatment in KPC mice effectively inhibited protein synthesis in vivo, resulting in a significant decrease in tumor kinetics in a short-term study and an improvement in survival in a longitudinal study. Reference 1. Chio II et al. NRF2 promotes tumor maintenance by modulating mRNA translation in pancreatic cancer. Cell 2016;166:963-76, doi:10.1016/j.cell.2016.06.056. Citation Format: Iok In Christine Chio, Christian Oertlin, Francis Robert, Karina Chan, Dana Kapellar Liberm, Abram Santana, Young Park, Ola Larsson, Jerry Pelletier, David Tuveson. Targeting redox dependencies in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA13.

Abstract A42: Bromodomain inhibitors suppress Nrf2-dependent HO-1 positive macrophage accumulation in murine models of KRAS-mutated pancreatic cancer

Abstract The Nrf2 (NF-E2-related factor-2) transcription factor regulates oxidative/xenobiotic stress responses and can drive cancer progression, metastasis and chemoresistance. Nrf2 also attenuates inflammation through elimination of reactive oxygen species (ROS) and regulation of macrophage specific genes involved in oxidative/stress responses. The tumor microenvironment (TME) in pancreatic cancer is rich in macrophages that promote cancer progression and chemoresistance. Tumor cells can use epigenetic modulation to evade immune recognition and to shape the TME toward an immunosuppressive phenotype. Bromodomain inhibitors are a class of drugs that target BET (bromodomain and extra-terminal) proteins, impairing their ability to bind to acetylated lysines and therefore interfering with transcriptional initiation and elongation. BET proteins regulate several genes responsible for cell growth, apoptosis and inflammation. Recent studies suggest that BET proteins may play a key role in the regulation of Nrf2-dependent antioxidative and inflammatory genes. Therefore, we hypothesized that the use of small BRD4 inhibitors could prevent the activation of Nrf2 –dependent macrophages in the pancreas. LSL-KrasG12D/+;Pdx-1-Cre (KC) and Nrf2 whole body knockout (KO) mice were stimulated with caerulein (acute protocol - 75 ug/kg injected i.p. every hour for 8 hours for 2 consecutive days) to induce pancreatitis. 72 hours later, macrophage infiltration into the pancreas was analyzed by flow cytometry. KC mice showed a significantly higher infiltration of macrophages when compared with Nrf2 KO or wild-type mice (51.1% vs 10.7% vs 21.5%). Treatment with I-BET 762 (60 mg/Kg BID once a day, starting 72 hours before caerulein stimulation and continued until the end of the study) did not reduce the infiltration of macrophages. However, I-BET 762 treatment did reduce the expression of HO-1, a downstream effector of Nrf2, as observed by Western blot and IHC. Nrf2 KO mice were almost devoid of HO-1 expression. Additionally, KC mice stimulated with caerulein and maintained on I-BET 762 treatment (60 mg/kg of diet) for 9 weeks showed a downregulation of CD206 expression in the pancreas, a marker of M2 macrophages. KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) mice will succumb to pancreatic cancer at an average of 23 weeks of age. I-BET 762 treatment for 12 weeks significantly reduced the levels of HO-1 expression in the pancreas microenvironment by 60% without altering the percentage of macrophages. HO-1 expression increased in RAW 267.4 cells or bone marrow-derived macrophages (BMDM) from wild-type mice stimulated with conditioned media from murine pancreatic cancer cells, but not in BMDM from Nrf2 KO mice. Moreover, the increase in HO-1 is decreased by treatment with I-BET 762 or N-acetyl-L-cysteine. This study suggests that bromodomain inhibitors can regulate Nrf2 activation in macrophages, with or without Kras mutations. Future studies will address the role of Nrf2 on macrophage function in vivo. Citation Format: Ana S. Leal, Karen T. Liby. Bromodomain inhibitors suppress Nrf2-dependent HO-1 positive macrophage accumulation in murine models of KRAS-mutated pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A42.

Undermining Glutaminolysis Bolsters Chemotherapy While NRF2 Promotes Chemoresistance in KRAS-Driven Pancreatic Cancers.

Pancreatic cancer is a disease with limited therapeutic options. Resistance to chemotherapies poses a significant clinical challenge for patients with pancreatic cancer and contributes to a high rate of recurrence. Oncogenic KRAS, a critical driver of pancreatic cancer, promotes metabolic reprogramming and upregulates NRF2, a master regulator of the antioxidant network. Here, we show that NRF2 contributed to chemoresistance and was associated with a poor prognosis in patients with pancreatic cancer. NRF2 activation metabolically rewired and elevated pathways involved in glutamine metabolism. This curbed chemoresistance in KRAS-mutant pancreatic cancers. In addition, manipulating glutamine metabolism restrained the assembly of stress granules, an indicator of chemoresistance. Glutaminase inhibitors sensitized chemoresistant pancreatic cancer cells to gemcitabine, thereby improving the effectiveness of chemotherapy. This therapeutic approach holds promise as a novel therapy for patients with pancreatic cancer harboring KRAS mutation. SIGNIFICANCE: These findings illuminate the mechanistic features of KRAS-mediated chemoresistance and provide a rationale for exploiting metabolic reprogramming in pancreatic cancer cells to confer therapeutic opportunities that could be translated into clinical trials. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/8/1630/F1.large.jpg.