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Purpose: In this single-institution phase II investigator-initiated study, we assessed the ability of MAPK and VEGF pathway blockade to overcome resistance to immunotherapy in microsatellite-stable metastatic colorectal cancer (MSS mCRC)., Patients and Methods: Patients with MSS, BRAF wild-type mCRC who progressed on ≥2 prior lines of therapy received pembrolizumab, binimetinib, and bevacizumab until disease progression or unacceptable toxicity. After a safety run-in, patients were randomized to a 7-day run-in of binimetinib or simultaneous initiation of all study drugs, to explore whether MEK inhibition may increase tumor immunogenicity. The primary endpoint was objective response rate (ORR) in all patients combined (by Response Evaluation Criteria in Solid Tumors v1.1)., Results: Fifty patients received study drug treatment; 54% were male with a median age of 55 years (range, 31-79). The primary endpoint, ORR, was 12.0% [95% confidence interval (CI) 4.5%-24.3%], which was not statistically different than the historical control data of 5% (P = 0.038, exceeding prespecified threshold of 0.025). The disease control rate was 70.0% (95% CI, 55.4%-82.1%), the median progression-free survival 5.9 months (95% CI, 4.2-8.7 months), and the median overall survival 9.3 months (95% CI, 6.7-12.2 months). No difference in efficacy was observed between the randomized cohorts. Grade 3 and 4 adverse events were observed in 56% and 8% of patients, respectively; the most common were rash (12%) and increased aspartate aminotransferase (12%)., Conclusions: Pembrolizumab, binimetinib, and bevacizumab failed to meet its primary endpoint of higher ORR compared with historical control data, demonstrated a high disease control rate, and demonstrated acceptable tolerability in refractory MSS mCRC., (©2024 The Authors; Published by the American Association for Cancer Research.)

Cancer cells exhibit rewired transcriptional regulatory networks that promote tumor growth and survival. However, the mechanisms underlying the formation of these pathological networks remain poorly understood. Through a pan-cancer epigenomic analysis, we found that primate-specific endogenous retroviruses (ERVs) are a rich source of enhancers displaying cancer-specific activity. In colorectal cancer and other epithelial tumors, oncogenic MAPK/AP1 signaling drives the activation of enhancers derived from the primate-specific ERV family LTR10. Functional studies in colorectal cancer cells revealed that LTR10 elements regulate tumor-specific expression of multiple genes associated with tumorigenesis, such as ATG12 and XRCC4 . Within the human population, individual LTR10 elements exhibit germline and somatic structural variation resulting from a highly mutable internal tandem repeat region, which affects AP1 binding activity. Our findings reveal that ERV-derived enhancers contribute to transcriptional dysregulation in response to oncogenic signaling and shape the evolution of cancer-specific regulatory networks.

Despite advancements in treating cutaneous melanoma, patients with acral and mucosal (A/M) melanomas still have limited therapeutic options and poor prognoses. We analyzed 156 melanomas (101 cutaneous, 28 acral, and 27 mucosal) using the Foundation One cancer-gene specific clinical testing platform and identified new, potentially targetable genomic alterations (GAs) in specific anatomic sites of A/M melanomas. Using novel pre-clinical models of A/M melanoma, we demonstrate that several GAs and corresponding oncogenic pathways associated with cutaneous melanomas are similarly targetable in A/M melanomas. Other alterations, including MYC and CRKL amplifications, were unique to A/M melanomas and susceptible to indirect targeting using the BRD4 inhibitor JQ1 or Src/ABL inhibitor dasatinib, respectively. We further identified new, actionable A/M-specific alterations, including an inactivating NF2 fusion in a mucosal melanoma responsive to dasatinib in vivo. Our study highlights new molecular differences between cutaneous and A/M melanomas, and across different anatomic sites within A/M, which may change clinical testing and treatment paradigms for these rare melanomas., (© 2024 UICC.)

Pancreatic ductal adenocarcinoma (PDAC) presents at advanced stages and is refractory to most treatment modalities. Wnt signaling activation plays a critical role in proliferation and chemotherapeutic resistance. Minimal media conditions, growth factor dependency, and Wnt dependency were determined via Wnt inhibition for seven patient derived organoids (PDOs) derived from pancreatic tumor organoid libraries (PTOL). Organoids demonstrating response in vitro were assessed in vivo using patient-derived xenografts. Wnt (in)dependent gene signatures were identified for each organoid. Panc269 demonstrated a trend of reduced organoid growth when treated with ETC-159 in combination with paclitaxel or gemcitabine as compared with chemotherapy or ETC-159 alone. Panc320 demonstrated a more pronounced anti-proliferative effect in the combination of ETC-159 and paclitaxel but not with gemcitabine. Panc269 and Panc320 were implanted into nude mice and treated with ETC-159, paclitaxel, and gemcitabine as single agents and in combination. The combination of ETC-159 and paclitaxel demonstrated an anti-tumor effect greater than ETC-159 alone. Extent of combinatory treatment effect were observed to a lesser extent in the Panc320 xenograft. Wnt (in)dependent gene signatures of Panc269 and 320 were consistent with the phenotypes displayed. Gene expression of several key Wnt genes assessed via RT-PCR demonstrated notable fold change following treatment in vivo. Each pancreatic organoid demonstrated varied niche factor dependencies, providing an avenue for targeted therapy, supported through growth analysis following combinatory treatment of Wnt inhibitor and standard chemotherapy in vitro. The clinical utilization of this combinatory treatment modality in pancreatic cancer PDOs has thus far been supported in our patient-derived xenograft models treated with Wnt inhibitor plus paclitaxel or gemcitabine. Gene expression analysis suggests there are key Wnt genes that contribute to the Wnt (in)dependent phenotypes of pancreatic tumors, providing plausible mechanistic explanation for Wnt (in)dependency and susceptibility or resistance to treatment on the genotypic level., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hawkins et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with a poor prognosis. Doxorubicin is part of standard curative therapy for TNBC, but chemotherapy resistance remains an important clinical challenge. Bocodepsin (OKI-179) is a small molecule class I histone deacetylase (HDAC) inhibitor that promotes apoptosis in TNBC preclinical models. The purpose of this study was to investigate the combination of bocodepsin and doxorubicin in preclinical TNBC models and evaluate the impact on terminal cell fate, including apoptosis and senescence., Methods: TNBC cell lines were treated with doxorubicin and CellTiter-Glo was used to assess proliferation and determine doxorubicin sensitivity. Select cell lines were treated with OKI-005 (in vitro version of bocodepsin) and doxorubicin and assessed for proliferation, apoptosis as measured by Annexin V/PI, and cell cycle by flow cytometry. Immunoblotting was used to assess changes in mediators of apoptosis, cell cycle arrest, and senescence. Senescence was measured by the senescence-associated β-galactosidase assay. An MDA-MB-231 xenograft in vivo model was treated with bocodepsin, doxorubicin, or the combination and assessed for inhibition of tumor growth. shRNA knockdown of p53 was performed in the CAL-51 cell line and proliferation, apoptosis and senescence were assessed in response to combination treatment., Results: OKI-005 and doxorubicin resulted in synergistic antiproliferative activity in TNBC cells lines regardless of p53 mutation status. The combination led to increased apoptosis and decreased senescence. In vivo, the combination resulted in increased tumor growth inhibition compared to either single agent. shRNA knock-down of p53 led to increased doxorubicin-induced senescence that was decreased with the addition of OKI-005 in vitro., Conclusion: The addition of bocodepsin to doxorubicin resulted in synergistic antiproliferative activity in vitro, improved tumor growth inhibition in vivo, and promotion of apoptosis which makes this a promising combination to overcome doxorubicin resistance in TNBC. Bocodepsin is currently in clinical development and has a favorable toxicity profile compared to other HDAC inhibitors supporting the feasibility of evaluating this combination in patients with TNBC., (© 2024. The Author(s).)

Single-cell technologies enable high-resolution studies of phenotype-defining molecular mechanisms. However, data sparsity and cellular heterogeneity make modeling biological variability across single-cell samples difficult. Here we present SCORPION, a tool that uses a message-passing algorithm to reconstruct comparable gene regulatory networks from single-cell/nuclei RNA-sequencing data that are suitable for population-level comparisons by leveraging the same baseline priors. Using synthetic data, we found that SCORPION outperformed 12 existing gene regulatory network reconstruction techniques. Using supervised experiments, we show that SCORPION can accurately identify differences in regulatory networks between wild-type and transcription factor-perturbed cells. We demonstrate SCORPION's scalability to population-level analyses using a single-cell RNA-sequencing atlas containing 200,436 cells from colorectal cancer and adjacent healthy tissues. The differences between tumor regions detected by SCORPION are consistent across multiple cohorts as well as with our understanding of disease progression, and elucidate phenotypic regulators that may impact patient survival., (© 2024. The Author(s).)

Defining feature of pancreatic ductal adenocarcinoma (PDAC) that participates in the high mortality rate and drug resistance is the immune-tolerant microenvironment which enables tumors to progress unabated by adaptive immunity. In this study, we report that PDAC cells release CSF-1 to induce nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) activation in myeloid cells. Increased NLRP3 expression was found in the pancreas of patients with PDAC when compared with normal pancreas which correlated with the formation of the NLRP3 inflammasome. Using human primary cells and an orthotopic PDAC mouse model, we show that NLRP3 activation is responsible for the maturation and release of the inflammatory cytokine IL1β which selectively drives Th2-type inflammation via COX2/PGE2 induction. As a result of this inflammation, primary tumors were characterized by reduced cytotoxic CD8+ T-cell activation and increased tumor expansion. Genetic deletion and pharmacologic inhibition of NLRP3 enabled the development of Th1 immunity, increased intratumoral levels of IL2, CD8+ T cell–mediated tumor suppression, and ultimately limited tumor growth. In addition, we observed that NLRP3 inhibition in combination with gemcitabine significantly increased the efficacy of the chemotherapy. In conclusion, this study provides a mechanism by which tumor-mediated NLRP3 activation exploits a distinct adaptive immunity response that facilitates tumor escape and progression. Considering the ability to block NLRP3 activity with safe and small orally active molecules, this protein represents a new promising target to improve the limited therapeutic options in PDAC., Significant: This study provides novel molecular insights on how PDAC cells exploit NLRP3 activation to suppress CD8 T-cell activation. From a translational perspective, we demonstrate that the combination of gemcitabine with the orally active NLRP3 inhibitor OLT1177 increases the efficacy of monotherapy., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, "humanized") mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2 -/- IL2RγC -/- NOD SIRPα ) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; "hot" tumors exhibit large immune infiltration while "cold" tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

Immune checkpoint inhibitors have been found to be effective in metastatic MSI-high colorectal cancers (CRC), however, have no efficacy in microsatellite stable (MSS) cancers, which comprise the majority of mCRC cases. Cabozantinib is a small molecule multi-tyrosine kinase inhibitor that is FDA approved in advanced renal cell, medullary thyroid, and hepatocellular carcinoma. Using Human Immune System (HIS) mice, we tested the ability of cabozantinib to prime MSS-CRC tumors to enhance the potency of immune checkpoint inhibitor nivolumab. In four independent experiments, we implanted distinct MSS-CRC patient-derived xenografts (PDXs) into the flanks of humanized BALB/c-Rag2 null Il2rγ null Sirpα NOD (BRGS) mice that had been engrafted with human hematopoietic stem cells at birth. For each PDX, HIS-mice cohorts were treated with vehicle, nivolumab, cabozantinib, or the combination. In three out of the four models, the combination had a lower tumor growth rate compared to vehicle or nivolumab-treated groups. Furthermore, interrogation of the HIS in immune organs and tumors by flow cytometry revealed increased Granzyme B+, TNFα+ and IFNγ+ CD4+ T cells among the human tumor infiltrating leukocytes (TIL) that correlated with reduced tumor growth in the combination-treated HIS-mice. Notably, slower growth correlated with increased expression of the CD4+ T cell ligand, HLA-DR, on the tumor cells themselves. Finally, the cabozantinib/nivolumab combination was tested in comparison to cobimetinib/atezolizumab. Although both combinations showed tumor growth inhibition, cabozantinib/nivolumab had enhanced cytotoxic IFNγ and TNFα+ T cells. This pre-clinical in vivo data warrants testing the combination in clinical trials for patients with MSS-CRC., Competing Interests: Exelixis supplied cabozantinib and funded this study, with TP as Principal Investigator, but had no role in study design, data collection, data analysis, data interpretation, or writing of the report. WM is the principal investigator of a phase II clinical trial of cabozantinib in patients with refractory metastatic colorectal cancer that is sponsored by Exelixis NCT03542877. AL is the principal investigator of a phase II clinical trial of cabozantinib in combination with nivolumab in metastatic colorectal cancer which is co-sponsored by Bristol Myers Squibb and Exelixis NCT04963283. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lang, Leal, Marín-Jiménez, Hartman, Shulman, Navarro, Lewis, Capasso, Bagby, Yacob, MacBeth, Freed, Eckhardt, Jordan, Blatchford, Pelanda, Lieu, Messersmith and Pitts.)

Background: AZD0156 is an oral inhibitor of ATM, a serine threonine kinase that plays a key role in DNA damage response (DDR) associated with double-strand breaks. Topoisomerase-I inhibitor irinotecan is used clinically to treat colorectal cancer (CRC), often in combination with 5-fluorouracil (5FU). AZD0156 in combination with irinotecan and 5FU was evaluated in preclinical models of CRC to determine whether low doses of AZD0156 enhance the cytotoxicity of irinotecan in chemotherapy regimens used in the clinic., Methods: Anti-proliferative effects of single-agent AZD0156, the active metabolite of irinotecan (SN38), and combination therapy were evaluated in 12 CRC cell lines. Additional assessment with clonogenic assay, cell cycle analysis, and immunoblotting were performed in 4 selected cell lines. Four colorectal cancer patient derived xenograft (PDX) models were treated with AZD0156, irinotecan, or 5FU alone and in combination for assessment of tumor growth inhibition (TGI). Immunofluorescence was performed on tumor tissues. The DDR mutation profile was compared across in vitro and in vivo models., Results: Enhanced effects on cellular proliferation and regrowth were observed with the combination of AZD0156 and SN38 in select models. In cell cycle analysis of these models, increased G2/M arrest was observed with combination treatment over either single agent. Immunoblotting results suggest an increase in DDR associated with irinotecan therapy, with a reduced effect noted when combined with AZD0156, which is more pronounced in some models. Increased TGI was observed with the combination of AZD0156 and irinotecan as compared to single-agent therapy in some PDX models. The DDR mutation profile was variable across models., Conclusions: AZD0156 and irinotecan provide a rational and active combination in preclinical colorectal cancer models. Variability across in vivo and in vitro results may be related to the variable DDR mutation profiles of the models evaluated. Further understanding of the implications of individual DDR mutation profiles may help better identify patients more likely to benefit from treatment with the combination of AZD0156 and irinotecan in the clinical setting., (© 2022. The Author(s).)

Histone deacetylases (HDACs) play critical roles in epigenomic regulation, and histone acetylation is dysregulated in many human cancers. Although HDAC inhibitors are active in T-cell lymphomas, poor isoform selectivity, narrow therapeutic indices, and a deficiency of reliable biomarkers may contribute to the lack of efficacy in solid tumors. In this article, we report the discovery and preclinical development of the novel, orally bioavailable, class-I-selective HDAC inhibitor, OKI-179. OKI-179 and its cell active predecessor OKI-005 are thioester prodrugs of the active metabolite OKI-006, a unique congener of the natural product HDAC inhibitor largazole. OKI-006, OKI-005, and subsequently OKI-179, were developed through a lead candidate optimization program designed to enhance physiochemical properties without eroding potency and selectivity relative to largazole. OKI-005 displays antiproliferative activity in vitro with induction of apoptosis and increased histone acetylation, consistent with target engagement. OKI-179 showed antitumor activity in preclinical cancer models with a favorable pharmacokinetic profile and on-target pharmacodynamic effects. Based on its potency, desirable class I HDAC inhibition profile, oral bioavailability, and efficacy against a broad range of solid tumors, OKI-179 is currently being evaluated in a first-in-human phase I clinical trial with plans for continued clinical development in solid tumor and hematologic malignancies., (©2021 American Association for Cancer Research.)

There is a clear need to identify targetable drivers of resistance and potential biomarkers for salvage therapy for patients with melanoma refractory to the combination of BRAF and MEK inhibition. In this study, we performed whole-exome sequencing on BRAF -V600E-mutant melanoma patient tumors refractory to the combination of BRAF/MEK inhibition and identified acquired oncogenic mutations in NRAS and loss of the tumor suppressor gene CDKN2A We hypothesized the acquired resistance mechanisms to BRAF/MEK inhibition were reactivation of the MAPK pathway and activation of the cell-cycle pathway, which can both be targeted pharmacologically with the combination of a MEK inhibitor (trametinib) and a CDK4/6 inhibitor (palbociclib). In vivo , we found that combination of CDK4/6 and MEK inhibition significantly decreased tumor growth in two BRAF/MEK inhibitor-resistant patient-derived xenograft models. In vitro , we observed that the combination of CDK4/6 and MEK inhibition resulted in synergy and significantly reduced cellular growth, promoted cell-cycle arrest, and effectively inhibited downstream signaling of MAPK and cell-cycle pathways in BRAF inhibitor-resistant cell lines. Knockdown of CDKN2A in BRAF inhibitor-resistant cells increased sensitivity to CDK4/6 inhibition alone and in combination with MEK inhibition. A key implication of our study is that the combination of CDK4/6 and MEK inhibitors overcomes acquired resistance to BRAF/MEK inhibitors, and loss of CDKN2A may represent a biomarker of response to the combination. Inhibition of the cell-cycle and MAPK pathway represents a promising strategy for patients with metastatic melanoma who are refractory to BRAF/MEK inhibitor therapy., (©2021 American Association for Cancer Research.)

Over the past decade, immunotherapies have revolutionized the treatment of cancer. Although the success of immunotherapy is remarkable, it is still limited to a subset of patients. More than 1500 clinical trials are currently ongoing with a goal of improving the efficacy of immunotherapy through co-administration of other agents. Preclinical, small-animal models are strongly desired to increase the pace of scientific discovery, while reducing the cost of combination drug testing in humans. Human immune system (HIS) mice are highly immune-deficient mouse recipients rtpeconstituted with human hematopoietic stem cells. These HIS-mice are capable of growing human tumor cell lines and patient-derived tumor xenografts. This model allows rapid testing of multiple, immune-related therapeutics for tumors originating from unique clinical samples. Using a cord blood-derived HIS-BALB/c-Rag2 null Il2rγ null SIRPα NOD (BRGS) mouse model, we summarize our experiments testing immune checkpoint blockade combinations in these mice bearing a variety of human tumors, including breast, colorectal, pancreatic, lung, adrenocortical, melanoma and hematological malignancies. We present in-depth characterization of the kinetics and subsets of the HIS in lymph and non-lymph organs and relate these to protocol development and immune-related treatment responses. Furthermore, we compare the phenotype of the HIS in lymph tissues and tumors. We show that the immunotype and amount of tumor infiltrating leukocytes are widely-variable and that this phenotype is tumor-dependent in the HIS-BRGS model. We further present flow cytometric analyses of immune cell subsets, activation state, cytokine production and inhibitory receptor expression in peripheral lymph organs and tumors. We show that responding tumors bear human infiltrating T cells with a more inflammatory signature compared to non-responding tumors, similar to reports of "responding" patients in human immunotherapy clinical trials. Collectively these data support the use of HIS mice as a preclinical model to test combination immunotherapies for human cancers, if careful attention is taken to both protocol details and data analysis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Marín-Jiménez, Capasso, Lewis, Bagby, Hartman, Shulman, Navarro, Yu, Rivard, Wang, Barkow, Geng, Kar, Yingst, Tufa, Dolan, Blatchford, Freed, Torres, Davila, Slansky, Pelanda, Eckhardt, Messersmith, Diamond, Lieu, Verneris, Wang, Kiseljak-Vassiliades, Pitts and Lang.)

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with high incidences of p53 mutations. AZD1775 (adavosertib, previously MK-1775) is a small molecule WEE1 inhibitor that abrogates the G2M checkpoint and can potentially synergize with DNA damaging therapies commonly used in PDAC treatment. The purpose of this study was to identify combination partners for AZD1775, including standard chemotherapy or targeted agents, in PDAC preclinical models. Low powered preliminary screens demonstrated that two of the four PDX models responded better to the combinations of AZD1775 with irinotecan or capecitabine than to either single agent. Following the screens, two full powered PDAC PDX models of differing p53 status were tested with the combinations of AZD1775 and irinotecan or capecitabine. The combinations of AZD1775 and SN38 or 5-FU were also tested on PDAC cell lines. Cellular proliferation was measured using an IncuCyte Live Cell Imager and apoptosis was measured using a Caspase-Glo 3/7 assay. Flow cytometry was conducted to measure alterations in cell cycle distribution. Western blot analysis was used to determine the effects of the drug combinations on downstream effectors. In PDX models with mutated p53 status, there was significant tumor growth inhibition from the combination of AZD1775 with irinotecan or capecitabine ( P ≤ 0.03), while PDX models with wild type p53 did not show anti-tumor synergy from the same combinations ( P ≥ 0.08). The combination of AZD1775 with SN38 or 5-FU significantly decreased proliferation in all PDAC cell lines, and enhanced apoptosis in multiple cell lines. Cell cycle distribution was disrupted from the combination of AZD1775 with SN38 or 5-FU which was recorded as G2M arrest and decreased G1 phase. AZD1775 inhibited phospho-CDC2 and increased the expression of γ H2AX that was either maintained or enhanced after combination with SN38 or 5-FU. The combination of AZD1775 with irinotecan/SN38 or capecitabine/5-FU showed anti-tumor effects in vivo and in vitro in PDAC models. These results support further investigation for these combination strategies to enhance outcomes for PDAC patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hartman, Bagby, Yacob, Simmons, MacBeth, Lieu, Davis, Leal, Tentler, Diamond, Eckhardt, Messersmith and Pitts.)

Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited systemic treatment options. RX-5902 is a novel anti-cancer agent that inhibits phosphorylated-p68 and thus attenuates nuclear β-catenin signaling. The purpose of this study was to evaluate the ability of β-catenin signaling blockade to enhance the efficacy of anti-CTLA-4 and anti-PD-1 immune checkpoint blockade in immunocompetent, preclinical models of TNBC., Methods: Treatment with RX-5902, anti-PD-1, anti-CTLA-4 or the combination was investigated in BALB/c mice injected with the 4 T1 TNBC cell line. Humanized BALB/c-Rag2 null Il2rγ null SIRPα NOD (hu-CB-BRGS) mice transplanted with a human immune system were implanted with MDA-MB-231 cells. Mice were randomized into treatment groups according to human hematopoietic chimerism and treated with RX-5902, anti-PD-1 or the combination. At sacrifice, bone marrow, lymph nodes, spleen and tumors were harvested for flow cytometry analysis of human immune cells., Results: The addition of RX-5902 to CTLA-4 or PD-1 inhibitors resulted in decreased tumor growth in the 4 T1 and human immune system and MDA-MB-231 xenograft models. Immunologic analyses demonstrated a significant increase in the number of activated T cells in tumor infiltrating lymphocytes (TILs) with RX-5902 treatment compared to vehicle (p < 0.05). In the RX-5902/nivolumab combination group, there was a significant increase in the percentage of CD4+ T cells in TILs and increased systemic granzyme B production (p < 0.01)., Conclusions: Conclusions: RX-5902 enhanced the efficacy of nivolumab in a humanized, preclinical model of TNBC. Several changes in immunologic profiles were noted in mice treated with RX-5902 and the combination, including an increase in activated TILs and a decrease in human myeloid populations, that are often associated with immunosuppression in a tumor microenvironment. RX-5902 also was shown to potentiate the effects of checkpoint inhibitors of CTLA4 and the PD-1 inhibitor in the 4 T-1 murine TNBC model. These findings indicate that RX-5902 may have important immunomodulatory, as well as anti-tumor activity, in TNBC when combined with a checkpoint inhibitor.

Chemoresistance to gemcitabine (GEM)-a frontline chemotherapeutic, resulting from its dysfunctional uptake and metabolism in cancer cells, is a major contributing factor for failed therapy in pancreatic cancer (PanC) patients. Therefore, there is an urgent need for agents that could reverse GEM resistance and allow continued chemosensitivity to the drug. We employed natural nontoxic agent (with anti-PanC potential) bitter melon juice (BMJ) and GEM to examine their combinatorial benefits against tumorigenesis of PanC patient-derived xenograft (PDX)-pancreatic ductal adenocarcinomas explants PDX272 (wild-type KRAS), PDX271 (mutant KRAS and SMAD4), and PDX266 (mutant KRAS). Anti-PanC efficacy of single agents vs combination in the three tumor explants, both at the end of active dosing regimen and following a drug-washout phase were compared. In animal studies, GEM alone treatment significantly inhibited PDX tumor growth, but effects were not sustained, as GEM-treated tumors exhibited regrowth posttreatment termination. However, combination-regimen displayed enhanced and sustained efficacy. Mechanistic assessments revealed that overcoming GEM resistance by coadministration with BMJ was possibly due to modulation of GEM transport/metabolism pathway molecules (ribonucleotide reductase regulatory subunit M1, human equilibrative nucleoside transporter 1, and deoxycytidine kinase). Study outcomes, highlighting significantly higher and sustained efficacy of GEM in combination with BMJ, make a compelling case for a clinical trial in PanC patients, wherein BMJ could be combined with GEM to target and overcome GEM resistance. In addition, given their specific effectiveness against KRAS-mutant tumors, this combination could be potentially beneficial to a broader PanC patient population., (© 2020 Wiley Periodicals LLC.)

Purpose: The purpose of this study was to evaluate the rational combination of TORC1/2 inhibitor TAK-228 and Aurora A kinase inhibitor alisertib in preclinical models of triple-negative breast cancer (TNBC) and to conduct a phase I dose escalation trial in patients with advanced solid tumors., Experimental Design: TNBC cell lines and patient-derived xenograft (PDX) models were treated with alisertib, TAK-228, or the combination and evaluated for changes in proliferation, cell cycle, mTOR pathway modulation, and terminal cellular fate, including apoptosis and senescence. A phase I clinical trial was conducted in patients with advanced solid tumors treated with escalating doses of alisertib and TAK-228 using a 3+3 design to determine the maximum tolerated dose (MTD)., Results: The combination of TAK-228 and alisertib resulted in decreased proliferation and cell-cycle arrest in TNBC cell lines. Treatment of TNBC PDX models resulted in significant tumor growth inhibition and increased apoptosis with the combination. In the phase I dose escalation study, 18 patients with refractory solid tumors were enrolled. The MTD was alisertib 30 mg b.i.d. days 1 to 7 of a 21-day cycle and TAK-228 2 mg daily, continuous dosing. The most common treatment-related adverse events were neutropenia, fatigue, nausea, rash, mucositis, and alopecia., Conclusions: The addition of TAK-228 to alisertib potentiates the antitumor activity of alisertib in vivo , resulting in increased cell death and apoptosis. The combination is tolerable in patients with advanced solid tumors and should be evaluated further in expansion cohorts with additional pharmacodynamic assessment., (©2020 American Association for Cancer Research.)

Current treatment for patients with metastatic melanoma include molecular-targeted therapies and immune checkpoint inhibitors. However, a subset of melanomas are difficult-to-treat. These melanomas include those without the genetic markers for targeted therapy, non-responsive to immunotherapy, and those who have relapsed or exhausted their therapeutic options. Therefore, it is necessary to understand and explore other biological processes that may provide new therapeutic approaches. One of most appealing is targeting the apoptotic/anti-apoptotic system that is effective against leukemia. We used genetic knockdown and pharmacologic approaches of BH3 mimetics to target anti-apoptotic BCL2 family members and identified MCL1 and BCLXL as crucial pro-survival members in melanoma. We then examined the effects of combining BH3 mimetics to target MCL1 and BCLXL in vitro and in vivo. These include clinical-trial-ready compounds such as ABT-263 (Navitoclax) and S63845/S64315 (MIK655). We used cell lines derived from patients with difficult-to-treat melanomas. In vitro, the combined inhibition of MCL1 and BCLXL resulted in significantly effective cell killing compared to single-agent treatment (p < 0.05) in multiple assays, including sphere assays. The combination-induced cell death was independent of BIM, and NOXA. Recapitulated in our mouse xenograft model, the combination inhibited tumor growth, reduced sphere-forming capacity (p < 0.01 and 0.05, respectively), and had tolerable toxicity (p > 0.40). Taken together, this study suggests that dual targeting of MCL1 and BCLXL should be considered as a treatment option for difficult-to-treat melanoma patients.

Triple-negative breast cancer (TNBC) is an aggressive subtype defined by lack of hormone receptor expression and non-amplified HER2. Adavosertib (AZD1775) is a potent, small-molecule, ATP-competitive inhibitor of the Wee1 kinase that potentiates the activity of many DNA-damaging chemotherapeutics and is currently in clinical development for multiple indications. The purpose of this study was to investigate the combination of AZD1775 and capecitabine/5FU in preclinical TNBC models. TNBC cell lines were treated with AZD1775 and 5FU and cellular proliferation was assessed in real-time using IncuCyte ® Live Cell Analysis. Apoptosis was assessed via the Caspase-Glo 3/7 assay system. Western blotting was used to assess changes in expression of downstream effectors. TNBC patient-derived xenograft (PDX) models were treated with AZD1775, capecitabine, or the combination and assessed for tumor growth inhibition. From the initial PDX screen, two of the four TNBC PDX models demonstrated a better response in the combination treatment than either of the single agents. As confirmation, two PDX models were expanded for statistical comparison. Both PDX models demonstrated a significant growth inhibition in the combination versus either of the single agents. (TNBC012, p < 0.05 combo vs. adavosertib or capecitabine, TNBC013, p < 0.01 combo vs. adavosertib or capecitabine.) An enhanced anti-proliferative effect was observed in the adavosertib/5FU combination treatment as measured by live cell analysis. An increase in apoptosis was observed in two of the four cell lines in the combination when compared to single-agent treatment. Treatment with adavosertib as a single agent resulted in a decrease in p-CDC2 in a dose-dependent manner that was also observed in the combination treatment. An increase in γH2AX in two of the four cell lines tested was also observed. No significant changes were observed in Bcl-xL following treatment in any of the cell lines. The combination of adavosertib and capecitabine/5FU demonstrated enhanced combination effects both in vitro and in vivo in preclinical models of TNBC. These results support the clinical investigation of this combination in patients with TNBC, including those with brain metastasis given the CNS penetration of both agents.

Context: Although the development of immune checkpoint inhibitors has transformed treatment strategies of several human malignancies, research models to study immunotherapy in adrenocortical carcinoma (ACC) are lacking., Objective: To explore the effect of anti-PD1 immunotherapy on the alteration of the immune milieu in ACC in a newly generated preclinical model and correlate with the response of the matched patient., Design, Setting, and Intervention: To characterize the CU-ACC2-M2B patient-derived xenograft in a humanized mouse model, evaluate the effect of a PD-1 inhibitor therapy, and compare it with the CU-ACC2 patient with metastatic disease., Results: Characterization of the CU-ACC2-humanized cord blood-BALB/c-Rag2nullIl2rγnullSirpaNOD model confirmed ACC origin and match with the original human tumor. Treatment of the mice with pembrolizumab demonstrated significant tumor growth inhibition (60%) compared with controls, which correlated with increased tumor infiltrating lymphocyte activity, with an increase of human CD8+ T cells (P < 0.05), HLA-DR+ T cells (P < 0.05) as well as Granzyme B+ CD8+ T cells (<0.001). In parallel, treatment of the CU-ACC2 patient, who had progressive disease, demonstrated a partial response with 79% to 100% reduction in the size of target lesions, and no new sites of metastasis. Pretreatment analysis of the patient's metastatic liver lesion demonstrated abundant intratumoral CD8+ T cells by immunohistochemistry., Conclusions: Our study reports the first humanized ACC patient-derived xenograft mouse model, which may be useful to define mechanisms and biomarkers of response and resistance to immune-based therapies, to ultimately provide more personalized care for patients with ACC., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

RX-5902 is a first-in-class anticancer agent targeting phosphorylated-p68 and attenuating nuclear shuttling of β-catenin. The purpose of this study was to evaluate the efficacy of RX-5902 in preclinical models of triple-negative breast cancer (TNBC) and to explore effects on β-catenin expression. A panel of 18 TNBC cell lines was exposed to RX-5902, and changes in proliferation, apoptosis, cellular ploidy, and effector protein expression were assessed. Gene expression profiling was used in sensitive and resistant cell lines with pathway analysis to explore pathways associated with sensitivity to RX-5902. The activity of RX-5902 was confirmed in vivo in cell line and patient-derived tumor xenograft (PDX) models. RX-5902 demonstrated potent antiproliferative activity in vitro against TNBC cell lines with an average IC 50 of 56 nmol/L in sensitive cell lines. RX-5902 treatment resulted in the induction of apoptosis, G 2 -M cell-cycle arrest, and aneuploidy in a subset of cell lines. RX-5902 was active in vivo against TNBC PDX models, and treatment resulted in a decrease in nuclear β-catenin. RX-5902 exhibited dose-proportional pharmacokinetics and plasma and tumor tissue in nude mice. Pathway analysis demonstrated an increase in the epithelial-to-mesenchymal transformation (EMT), TGFβ, and Wnt/β-catenin pathways associated with sensitivity to RX-5902. RX-5902 is active against in vitro and in vivo preclinical models of TNBC. Target engagement was confirmed with decreases in nuclear β-catenin and MCL-1 observed, confirming the proposed mechanism of action. This study supports the continued investigation of RX-5902 in TNBC and combinations with immunotherapy., (©2019 American Association for Cancer Research.)

Adrenocortical carcinoma (ACC) is an aggressive orphan malignancy with less than 35% 5-year survival and 75% recurrence. Surgery remains the primary therapy and mitotane, an adrenolytic, is the only FDA-approved drug with wide-range toxicities and poor tolerability. There are no targeted agents available to date. For the last three decades, H295R cell line and its xenograft were the only available preclinical models. We recently developed two new ACC patient-derived xenograft mouse models and corresponding cell lines (CU-ACC1 and CU-ACC2) to advance research in the field. Here, we have utilized these novel models along with H295R cells to establish the mitotic PDZ-binding kinase (PBK) as a promising therapeutic target. PBK is overexpressed in ACC samples and correlates with poor survival. We show that PBK is regulated by FOXM1 and targeting PBK via shRNA decreased cell proliferation, clonogenicity and anchorage-independent growth in ACC cell lines. PBK silencing inhibited pAkt, pp38MAPK and pHistone H3 altering the cell cycle. Therapeutically, targeting PBK with the small-molecule inhibitor HITOPK032 phenocopied PBK-specific modulation of pAkt and pHistone H3, but also induced apoptosis via activation of JNK. Consistent with in vitro findings, treatment of CU-ACC1 PDXs with HITOPK032 significantly reduced tumor growth by 5-fold (P < 0.01). Treated tumor tissues demonstrated increased rates of apoptosis and JNK activation, with decreased pAkt and Histone H3 phosphorylation, consistent with effects observed in ACC cell lines. Together these studies elucidate the mechanism of PBK in ACC tumorigenesis and establish the potential therapeutic potential of HITOPK032 in ACC patients.

Background: The success of agents that reverse T-cell inhibitory signals, such as anti-PD-1/PD-L1 therapies, has reinvigorated cancer immunotherapy research. However, since only a minority of patients respond to single-agent therapies, methods to test the potential anti-tumor activity of rational combination therapies are still needed. Conventional murine xenograft models have been hampered by their immune-compromised status; thus, we developed a hematopoietic humanized mouse model, hu-CB-BRGS, and used it to study anti-tumor human immune responses to triple-negative breast cancer (TNBC) cell line and patient-derived colorectal cancer (CRC) xenografts (PDX)., Methods: BALB/c-Rag2 null Il2rγ null SIRPα NOD (BRGS) pups were humanized through transplantation of cord blood (CB)-derived CD34+ cells. Mice were evaluated for human chimerism in the blood and assigned into experimental untreated or nivolumab groups based on chimerism. TNBC cell lines or tumor tissue from established CRC PDX models were implanted into both flanks of humanized mice and treatments ensued once tumors reached a volume of ~150mm 3 . Tumors were measured twice weekly. At end of study, immune organs and tumors were collected for immunological assessment., Results: Humanized PDX models were successfully established with a high frequency of tumor engraftment. Humanized mice treated with anti-PD-1 exhibited increased anti-tumor human T-cell responses coupled with decreased Treg and myeloid populations that correlated with tumor growth inhibition. Combination therapies with anti-PD-1 treatment in TNBC-bearing mice reduced tumor growth in multi-drug cohorts. Finally, as observed in human colorectal patients, anti-PD-1 therapy had a strong response to a microsatellite-high CRC PDX that correlated with a higher number of human CD8+ IFNγ+ T cells in the tumor., Conclusion: Hu-CB-BRGS mice represent an in vivo model to study immune checkpoint blockade to human tumors. The human immune system in the mice is inherently suppressed, similar to a tumor microenvironment, and thus allows growth of human tumors. However, the suppression can be released by anti-PD-1 therapies and inhibit tumor growth of some tumors. The model offers ample access to lymph and tumor cells for in-depth immunological analysis. The tumor growth inhibition correlates with increased CD8 IFNγ+ tumor infiltrating T cells. These hu-CB-BRGS mice provide a relevant preclinical animal model to facilitate prioritization of hypothesis-driven combination immunotherapies.

Oncogenic BRAF fusions have emerged as an alternate mechanism for BRAF activation in melanomas and other cancers. A number of BRAF fusions with different 5' gene partners and BRAF exon breakpoints have been described, but the effects of different partners and breakpoints on cancer phenotypes and treatment responses has not been well characterized. Targeted RNA sequencing was used to screen 60 melanoma patient-derived xenograft (PDX) models for BRAF fusions. We identified three unique BRAF fusions, including a novel SEPT3-BRAF fusion, occurring in four tumors (4/60, 6.7%), all of which were "pan-negative" (lacking other common mutations) (4/18, 22.2%). The BRAF fusion PDX models showed variable growth rates and responses to MAPK inhibitors in vivo. Overexpression of BRAF fusions identified in our study, as well as other BRAF fusions previously identified in melanomas, resulted in a high degree of variability in 2D proliferation and 3D invasion between the different fusions. While exogenously expressed BRAF fusions all responded to MAPK inhibition in vitro, we observed potential differences in signaling and feedback mechanisms. In summary, BRAF fusions are actionable therapeutic targets, however there are significant differences in phenotypes, treatment responses, and signaling which may be clinically relevant.

Molecularly-targeted agents have improved outcomes for a subset of patients with BRAF -mutated melanoma, but treatment of resistant and BRAF wild-type tumors remains a challenge. The MERTK receptor tyrosine kinase is aberrantly expressed in melanoma and can contribute to oncogenic phenotypes. Here we report the effect of treatment with a MERTK-selective small molecule inhibitor, UNC2025, in preclinical models of melanoma. In melanoma cell lines, treatment with UNC2025 potently inhibited phosphorylation of MERTK and downstream signaling, induced cell death, and decreased colony formation. In patient-derived melanoma xenograft models, treatment with UNC2025 blocked or significantly reduced tumor growth. Importantly, UNC2025 had similar biochemical and functional effects in both BRAF -mutated and BRAF wild-type models and irrespective of NRAS mutational status, implicating MERTK inhibition as a potential therapeutic strategy in tumors that are not amenable to BRAF-targeting and for which there are limited treatment options. In BRAF -mutated cell lines, combined treatment with UNC2025 and the BRAF inhibitor vemurafenib provided effective inhibition of oncogenic signaling through ERK, AKT, and STAT6, increased induction of cell death, and decreased colony-forming potential. Similarly, in NRAS -mutated cell lines, addition of UNC2025 to cobimetinib therapy increased cell death and decreased colony-forming potential. In a BRAF -mutated patient-derived xenograft, treatment with combined UNC2025 and vemurafenib was well-tolerated and significantly decreased tumor growth compared with vemurafenib alone. These data support the use of UNC2025 for treatment of melanoma, irrespective of BRAF or NRAS mutational status, and suggest a role for MERTK and targeted combination therapy in BRAF and NRAS -mutated melanoma., (©2018 American Association for Cancer Research.)

Cancer is a disease caused by several factors characterized by uncontrolled cell division, growth, and survival. ENMD-2076, is a novel orally active small molecule multikinase inhibitor targeting angiogenesis, proliferation, and the cell cycle. It is selectively active against the mitotic kinases aurora A and B, and kinases responsible for angiogenesis including VEGFR2/KDR and FGFR1 and 2. ENMD-2076 has been shown to inhibit tumor growth and prevent angiogenesis in vitro and in vivo in preclinical cancer models. Moreover, in a phase I trial, ENMD-2076 was well tolerated, exhibited a linear pharmacokinetic profile, and showed a promising antitumor activity in a number of solid tumors. In this study, we show that ENMD-2076 has antiproliferative effects, causes cell cycle arrest, and has activity in preclinical models of colorectal cancer (CRC), including patient-derived xenograft (PDX) models. Forty-seven human CRC cell lines were exposed in vitro to ENMD-2076 and analyzed for effects on cell cycle, apoptosis, and downstream effector proteins. The drug was then tested against 20 human CRC PDX models to further evaluate in-vivo antitumor activity. We show that ENMD-2076 exhibits a broad range of activity against a large panel of CRC cell lines with varying molecular characteristics. Mechanistically, ENMD-2076 exposure resulted in a G2/M cell cycle arrest, an increase in aneuploidy, and cell death in responsive cell lines. In addition, ENMD-2076 treatment resulted in a promising antitumor activity in CRC PDX models. These results support the continued development of ENMD-2076 in CRC including further exploration of rational combinations.

Antiangiogenic therapy used in treatment of metastatic colorectal cancer (mCRC) inevitably succumbs to treatment resistance. Upregulation of MET may play an essential role to acquired anti-VEGF resistance. We previously reported that cabozantinib (XL184), an inhibitor of receptor tyrosine kinases (RTK) including MET, AXL, and VEGFR2, had potent antitumor effects in mCRC patient-derived tumor explant models. In this study, we examined the mechanisms of cabozantinib sensitivity, using regorafenib as a control. The tumor growth inhibition index (TGII) was used to compare treatment effects of cabozantinib 30 mg/kg daily versus regorafenib 10 mg/kg daily for a maximum of 28 days in 10 PDX mouse models. In vivo angiogenesis and glucose uptake were assessed using dynamic contrast-enhanced (DCE)-MRI and [ 18 F]-FDG-PET imaging, respectively. RNA-Seq, RTK assay, and immunoblotting analysis were used to evaluate gene pathway regulation in vivo and in vitro Analysis of TGII demonstrated significant antitumor effects with cabozantinib compared with regorafenib (average TGII 3.202 vs. 48.48, respectively; P = 0.007). Cabozantinib significantly reduced vascularity and glucose uptake compared with baseline. Gene pathway analysis showed that cabozantinib significantly decreased protein activity involved in glycolysis and upregulated proteins involved in autophagy compared with control, whereas regorafenib did not. The combination of two separate antiautophagy agents, SBI-0206965 and chloroquine, plus cabozantinib increased apoptosis in vitro Cabozantinib demonstrated significant antitumor activity, reduction in tumor vascularity, increased autophagy, and altered cell metabolism compared with regorafenib. Our findings support further evaluation of cabozantinib and combinational approaches targeting autophagy in colorectal cancer. Mol Cancer Ther; 17(10); 2112-22. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Adrenocortical cancer (ACC) is an orphan malignancy that results in heterogeneous clinical phenotypes and molecular genotypes. There are no curative treatments for this deadly cancer with 35% survival at five years. Our understanding of the underlying pathobiology and our ability to test novel therapeutic targets has been limited due to the lack of preclinical models. Here, we report the establishment of two new ACC cell lines and corresponding patient-derived xenograft (PDX) models. CU-ACC1 cell line and PDX were derived from a perinephric metastasis in a patient whose primary tumor secreted aldosterone. CU-ACC2 cell line and PDX were derived from a liver metastasis in a patient with Lynch syndrome. Short tandem repeat profiling confirmed consistent matches between human samples and models. Both exomic and RNA sequencing profiling were performed on the patient samples and the models, and hormonal secretion was evaluated in the new cell lines. RNA sequencing and immunohistochemistry confirmed the expression of adrenal cortex markers in the PDXs and human tumors. The new cell lines replicate two of the known genetic models of ACC. CU-ACC1 cells had a mutation in CTNNB1 and secreted cortisol but not aldosterone. CU-ACC2 cells had a TP53 mutation and loss of MSH2 consistent with the patient's known germline mutation causing Lynch syndrome. Both cell lines can be transfected and transduced with similar growth rates. These new preclinical models of ACC significantly advance the field by allowing investigation of underlying molecular mechanisms of ACC and the ability to test patient-specific therapeutic targets., (© 2018 Society for Endocrinology.)

Background: Polo-like kinase 1 (Plk1) is a serine/threonine kinase that is a key regulator of multiple stages of mitotic progression. Plk1 is upregulated in many tumor types including colorectal cancer (CRC) and portends a poor prognosis. TAK-960 is an ATP-competitive Plk1 inhibitor that has demonstrated efficacy across a broad range of cancer cell lines, including CRC. In this study, we investigated the activity of TAK-960 against a large collection of CRC models including 55 cell lines and 18 patient-derived xenografts., Methods: Fifty-five CRC cell lines and 18 PDX models were exposed to TAK-960 and evaluated for proliferation (IC 50 ) and Tumor Growth Inhibition Index, respectively. Additionally, 2 KRAS wild type and 2 KRAS mutant PDX models were treated with TAK-960 as single agent or in combination with cetuximab or irinotecan. TAK-960 mechanism of action was elucidated through immunoblotting and cell cycle analysis., Results: CRC cell lines demonstrated a variable anti-proliferative response to TAK-960 with IC 50 values ranging from 0.001 to > 0.75 μmol/L. Anti-proliferative effects were sustained after removal of drug. Following TAK-960 treatment a highly variable accumulation of mitotic (indicating cell cycle arrest) and apoptotic markers was observed. Cell cycle analysis demonstrated that TAK-960 treatment induced G2/M arrest and polyploidy. Six out of the eighteen PDX models responded to single agent TAK-960 therapy (TGII< 20). The addition of TAK-960 to standard of care chemotherapy resulted in largely additive antitumor effects., Conclusion: TAK-960 is an active anti-proliferative agent against CRC cell lines and PDX models. Collectively, these data suggest that TAK-960 may be of therapeutic benefit alone or in combination with other agents, although future work should focus on the development of predictive biomarkers and hypothesis-driven rational combinations.

Background: Antibody-drug conjugates (ADCs) are an emerging technology consisting of an antibody, linker, and toxic agent, which have the potential to offer a targeted therapeutic approach. A novel target recently explored for the treatment of pancreatic cancer is guanylyl cyclase C (GCC). The objective of this study was to determine the anti-tumorigenic activity of TAK-264, an investigational ADC consisting of an antibody targeting GCC linked to a monomethyl auristatin E payload via a peptide linker., Methods: The antiproliferative effects of TAK-264 assessed in a panel of eleven pancreatic cancer cell lines. Additionally, ten unique pancreatic ductal adenocarcinoma cancer patient-derived xenograft models were treated with TAK-264 and the efficacy was determined. Baseline levels of GCC were analyzed on PDX models and cell lines. Immunoblotting was performed to evaluate the effects of TAK-264 on downstream effectors., Results: GCC protein expression was analyzed by immunoblotting in both normal and tumor tissue; marked increase in GCC expression was observed in tumor tissue. The in vitro experiments demonstrated a range of responses to TAK-264. Eight of the ten PDAC PDX models treated with TAK-264 demonstrated a statistically significant tumor growth inhibition. Immunoblotting demonstrated an increase in phosphorylated-HistoneH3 in both responsive and less responsive cell lines and PDAC PDX models treated with TAK-264. There was no correlation between baseline levels of GCC and response in either PDX or cell line models., Conclusion: TAK-264 has shown suppression activity in pancreatic cancer cell lines and in pancreatic PDX models. These findings support further investigation of ADC targeting GCC., Competing Interests: CONFLICT OF INTEREST Potential conflict of interest are authors TK, BS, HD, TC who are employees of Takeda Limited. Takeda Pharmaceuticals partially funded this project and had no role in study design and decision to publish. The funders had no role in data collection and analysis or preparation of the manuscript. Authors ARS, AN, SMB, JJA, BY, KQ, JLG, WAM, TMP have no conflicts of interest.

Oncogenic ALK fusions occur in several types of cancer and can be effectively treated with ALK inhibitors; however, ALK fusions and treatment response have not been characterized in malignant melanomas. Recently, a novel isoform of ALK ( ALK ATI ) was reported in 11% of melanomas but the response of melanomas expressing ALK ATI to ALK inhibition has not been well characterized. We analyzed 45 melanoma patient-derived xenograft models for ALK mRNA and protein expression. ALK expression was identified in 11 of 45 (24.4%) melanomas. Ten melanomas express wild-type (wt) ALK and/or ALK ATI and one mucosal melanoma expresses multiple novel EML4-ALK fusion variants. Melanoma cells expressing different ALK variants were tested for response to ALK inhibitors. Whereas the melanoma expressing EML4-ALK were sensitive to ALK inhibitors in vitro and in vivo , the melanomas expressing wt ALK or ALK ATI were not sensitive to ALK inhibitors. In addition, a patient with mucosal melanoma expressing ALK ATI was treated with an ALK/ROS1/TRK inhibitor (entrectinib) on a phase I trial but did not respond. Our results demonstrate ALK fusions occur in malignant melanomas and respond to targeted therapy, whereas melanomas expressing ALK ATI do not respond to ALK inhibitors. Targeting ALK fusions is an effective therapeutic option for a subset of melanoma patients, but additional clinical studies are needed to determine the efficacy of targeted therapies in melanomas expressing wt ALK or ALK ATI Mol Cancer Ther; 17(1); 222-31. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Irinotecan, a standard of care therapy for CRC, elicits cytotoxic effects by generating double strand breaks resulting in DNA damage. The activation of the ATM pathway plays a fundamental role in regulating the cellular response and repair to DNA damage. The objective of this preclinical study was to determine whether ATM inhibition would enhance sensitivity to irinotecan treatment. Treatment effects of AZ31, irinotecan or AZ31 + irinotecan were investigated in CRC cell lines and CRC patient derived xenografts. Activation of ATM and downstream targets p-RAD50 and p-H2AX were evaluated by immunohistochemistry. Combinational effects were demonstrated in 4 out of 8 CRC explants. Interestingly, each of the combinational sensitive CRC PDX models were shown to be more resistant to irinotecan single agent therapy. Treatment with irinotecan significantly elevated the ATM pathway evident by an increase in the activation of H2AX and RAD50. Combinational therapy reduced the activation of H2AX and RAD50 when compared to irinotecan alone in the combination sensitive CRC098. AZ31 + irinotecan was effective at reducing tumor growth in tumors that exhibited resistance to irinotecan in our CRC PDX model. These findings support further investigation of this combinational therapy for the treatment of CRC patients., Competing Interests: CONFLICTS OF INTEREST None.

Human patient-derived xenograft (PDX) tumors, propagated in immunodeficient mice, are rapidly growing in use as a model for cancer research. Horizontal transfer between mice, without in vitro cell culture, allows these tumors to retain many of their unique characteristics from their individual patient of origin. However, the immunodeficient mouse strains used to grow these tumors are susceptible to numerous opportunistic pathogens, including Corynebacterium bovis. At our institution, 2 in vivo tumor banks of PDX tumors had been maintained within nude mouse colonies enzootically infected with C. bovis. Elimination of C. bovis from these colonies required the aseptic harvest and horizontal transfer of tumor tissue between infected and naïve recipient mice without cross-contamination. Out of necessity, we developed a standard operating procedure using enhancements to traditional aseptic surgical technique with concurrent application of both procedural and physical barriers to prevent C. bovis transmission. By using these methods, all 61 unique PDX tumor models were successfully harvested from C. bovis-infected mice and transferred into recipient mice without transmission of infection. Our data demonstrate that, in situations where C. bovis-free colonies can be established and maintained, this procedure can successfully be used to eliminate C. bovis from an in vivo tumor bank of valuable PDX tumors.

Background The neddylation pathway conjugates NEDD8 to cullin-RING ligases and controls the proteasomal degradation of specific proteins involved in essential cell processes. Pevonedistat (MLN4924) is a selective small molecule targeting the NEDD8-activating enzyme (NAE) and inhibits an early step in neddylation, resulting in DNA re-replication, cell cycle arrest and death. We investigated the anti-tumor potential of pevonedistat in preclinical models of melanoma. Methods Melanoma cell lines and patient-derived tumor xenografts (PDTX) treated with pevonedistat were assessed for viability/apoptosis and tumor growth, respectively, to identify sensitive/resistant models. Gene expression microarray and gene set enrichment analyses were performed in cell lines to determine the expression profiles and pathways of sensitivity/resistance. Pharmacodynamic changes in treated-PDTX were also characterized. Results Pevonedistat effectively inhibited cell viability (IC 50  < 0.3 μM) and induced apoptosis in a subset of melanoma cell lines. Sensitive and resistant cell lines exhibited distinct gene expression profiles; sensitive models were enriched for genes involved in DNA repair, replication and cell cycle regulation, while immune response and cell adhesion pathways were upregulated in resistant models. Pevonedistat also reduced tumor growth in melanoma cell line xenografts and PDTX with variable responses. An accumulation of pevonedistat-NEDD8 adduct and CDT1 was observed in sensitive tumors consistent with its mechanism of action. Conclusions This study provided preclinical evidence that NAE inhibition by pevonedistat has anti-tumor activity in melanoma and supports the clinical benefits observed in recent Phase 1 trials of this drug in melanoma patients. Further investigations are warranted to develop rational combinations and determine predictive biomarkers of pevonedistat.

Background: The Aurora kinases are a family of serine/threonine kinases comprised of Aurora A, B, and C which execute critical steps in mitotic and meiotic progression. Alisertib (MLN8237) is an investigational Aurora A selective inhibitor that has demonstrated activity against a wide variety of tumor types in vitro and in vivo, including CRC., Results: CRC cell lines demonstrated varying sensitivity to alisertib with IC50 values ranging from 0.06 to > 5 umol/L. Following exposure to alisertib we observed a decrease in pAurora A, B and C in four CRC cell lines. We also observed an increase in p53 and p21 in a sensitive p53 wildtype cell line in contrast to the p53 mutant cell line or the resistant cell lines. The addition of alisertib to standard CRC treatments demonstrated improvement over single agent arms; however, the benefit was largely less than additive, but not antagonistic., Methods: Forty-seven CRC cell lines were exposed to alisertib and IC50s were calculated. Twenty-one PDX models were treated with alisertib and the Tumor Growth Inhibition Index was assessed. Additionally, 5 KRAS wildtype and mutant PDX models were treated with alisertib as single agent or in combination with cetuximab or irinotecan, respectively., Conclusion: Alisertib demonstrated anti-proliferative effects against CRC cell lines and PDX models. Our data suggest that the addition of alisertib to standard therapies in colorectal cancer if pursued clinically, will require further investigation of patient selection strategies and these combinations may facilitate future clinical studies., Competing Interests: JE and ELB are both currently employed by Millennium Pharmaceuticals, INC., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited.

Background: CRC is a significant cause of cancer mortality, and new therapies are needed for patients with advanced disease. TAK-733 is a highly potent and selective investigational novel MEK allosteric site inhibitor., Materials and Methods: In a preclinical study of TAK-733, a panel of CRC cell lines were exposed to varying concentrations of the agent for 72 hours followed by a sulforhodamine B assay. Twenty patient-derived colorectal cancer xenografts were then treated with TAK-733 in vivo. Tumor growth inhibition index (TGII) was assessed to evaluate the sensitivity of the CRC explants to TAK-733 while linear regression was utilized to investigate the predictive effects of genotype on the TGII of explants., Results: Fifty-four CRC cell lines were exposed to TAK-733, while 42 cell lines were deemed sensitive across a broad range of mutations. Eighty-two percent of the cell lines within the sensitive subset were BRAF or KRAS/NRAS mutant, whereas 80% of the cell lines within the sensitive subset were PIK3CA WT. Twenty patient-derived human tumor CRC explants were then treated with TAK-733. In total, 15 primary human tumor explants were found to be sensitive to TAK-733 (TGII ≤ 20%), including 9 primary human tumor explants that exhibited tumor regression (TGII > 100%). Explants with a BRAF/KRAS/NRAS mutant and PIK3CA wild-type genotype demonstrated increased sensitivity to TAK-733 with a median TGII of -6%. MEK-response gene signatures also correlated with responsiveness to TAK-733 in KRAS-mutant CRC., Conclusions: The MEK inhibitor TAK-733 demonstrated robust antitumor activity against CRC cell lines and patient-derived tumor explants. While the preclinical activity observed in this study was considerable, single-agent efficacy in the clinic has been limited in CRC, supporting the use of these models in an iterative manner to elucidate resistance mechanisms that can guide rational combination strategies.

Aurora A kinase and MEK inhibitors induce different, and potentially complementary, effects on the cell cycle of malignant cells, suggesting a rational basis for utilizing these agents in combination. In this work, the combination of an Aurora A kinase and MEK inhibitor was evaluated in pre-clinical colorectal cancer models, with a focus on identifying a subpopulation in which it might be most effective. Increased synergistic activity of the drug combination was identified in colorectal cancer cell lines with concomitant KRAS and PIK3CA mutations. Anti-proliferative effects were observed upon treatment of these double-mutant cell lines with the drug combination, and tumor growth inhibition was observed in double-mutant human tumor xenografts, though effects were variable within this subset. Additional evaluation suggests that degree of G2/M delay and p53 mutation status affect apoptotic activity induced by combination therapy with an Aurora A kinase and MEK inhibitor in KRAS and PIK3CA mutant colorectal cancer. Overall, in vitro and in vivo testing was unable to identify a subset of colorectal cancer that was consistently responsive to the combination of a MEK and Aurora A kinase inhibitor.

Background: The activation of the MAPK and PI3K/AKT/mTOR pathways is implicated in the majority of cancers. Activating mutations in both of these pathways has been described in colorectal cancer (CRC), thus indicating their potential as therapeutic targets. This study evaluated the combination of a PI3K/mTOR inhibitor (PF-04691502/PF-502) in combination with a MEK inhibitor (PD-0325901/PD-901) in CRC cell lines and patient-derived CRC tumor xenograft models (PDTX)., Materials and Methods: The anti-proliferative effects of PF-502 and PD-901 were assessed as single agents and in combination against a panel of CRC cell lines with various molecular backgrounds. Synergy was evaluated using the Bliss Additivity method. In selected cell lines, we investigated the combination effects on downstream effectors by immunoblotting. The combination was then evaluated in several fully genetically annotated CRC PDTX models., Results: The in vitro experiments demonstrated a wide range of IC50 values for both agents against a cell line panel. The combination of PF-502 and PD-901 demonstrated synergistic anti-proliferative activity with Bliss values in the additive range. As expected, p-AKT and p-ERK were downregulated by PF-502 and PD-901, respectively. In PDTX models, following a 30-day exposure to PF-502, PD-901 or the combination, the combination demonstrated enhanced reduction in tumor growth as compared to either single agent regardless of KRAS or PI3K mutational status., Conclusions: The combination of a PI3K/mTOR and a MEK inhibitor demonstrated enhanced anti-proliferative effects against CRC cell lines and PDTX models.