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Background: Triple-negative breast cancer (TNBC) is an aggressive subtype defined by lack of hormone receptor expression and non-amplified HER2. TNBC accounts for approximately 15% of breast cancer cases, however, is associated with an increased risk of cancer recurrence, brain metastasis, and death due to metastatic breast cancer. Mutations in p53 are common in TNBC, occurring in approximately 85% of tumors. While a number of promising targeted therapies are on the horizon in TNBC including immunotherapy, there remains an unmet need for active targeted therapies where chemotherapy remains the standard treatment for metastatic disease and results in a median survival of 12-18 months. 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. AZD1775 potentiates the activity of DNA-damaging and antimetabolite chemotherapeutics in preclinical models without TP53-deficiency, possibly due to baseline replicative stress or compromised DNA repair proficiency. A previous unbiased screen of CTEP compounds in TNBC PDX models demonstrated that the combination of adavosertib and capecitabine/5FU had greater anti-proliferative effects than either of the single agents. The purpose of this study was to further investigate the combination of adavosertib and capecitabine/5FU in preclinical TNBC models. Methods: HCC1937, CAL51, MDA-MB-231 and MDA-MB-468 cells were plated in 96-well plates and exposed to increasing concentrations of adavosertib, 5FU, or the combination. 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 CDC2, phospho-CDC2, H2AX, and Bcl-xL. TNBC PDX models CU_TNBC_012 and CU_TNBC_013 were treated with vehicle, adavosertib, capecitabine, or the combination and assessed for tumor growth inhibition. Results: 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 Citation Format: Todd M Pitts, Dennis M Simmons, Kyrie Dailey, Stacey M Bagby, Sarah J Hartman, Betelehem W Yacob, Brian Gittleman, John J Tentler, Diana Cittely, D. Ryan Ormond, Wells A Messersmith, S Gail Eckhardt, Jennifer R Diamond. Wee1 inhibition enhances the anti-tumor effects of capecitabine in preclinical models of triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-25.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype distinguished by its lack of expression of receptors for estrogen, progesterone, and normal human epidermal growth factor 2 expression. TNBC is difficult to treat and is associated with a high risk of recurrence and mortality. In order to effectively treat TNBC, alternative therapeutic targets need to be identified. Wee1 is a tyrosine kinase that phosphorylates CDC2 to pause the cell cycle at the G2 checkpoint as a means to delay mitosis while DNA damage undergoes repair. Inactivation of Wee1 via adavosertib (AZD1775, MK1775), a highly selective inhibitor of Wee1, allows CDC25 to dephosphorylate the CDC2/cyclin B complex resulting in premature initiation of mitosis and, ultimately, mitotic catastrophe and apoptosis. An unbiased screen of adovosertib in combination with other targeted compounds in TNBC patient-derived xenograft (PDX) models demonstrated that the combination of adavosertib and navitoclax, an inhibitor of anti-apoptotic BCL-2 and BCL-XL proteins, had greater efficacy than the single agents alone. The purpose of this study was to investigate the combination of adavosertib and navitoclax in preclinical TNBC models, both in vitro and in vivo. Methods: HCC1937, CAL51, MDA-MB-231 and MDA-MB-468 cells were plated in 96-well plates and exposed to increasing concentrations of adavosertib (125nM – 1000nM), navitoclax (2500nM – 10000nM), or the combination. Cellular proliferation was assessed in real-time using IncuCyte Live Cell Analysis, followed by endpoint sulforhodamine B (SRB) assay. Combination effects were analyzed using Calcusyn to determine combination indexes (CI). Apoptosis was assessed via the Caspase 3/7 assay. Western blotting was used to assess changes in expression of CDC2, phospho-CDC2, and BCL2. TNBC PDX models CU_TNBC_013 and CU_TNBC_014 were treated with vehicle, adavosertib (50mg/kg), navitoclax (100mg/kg), or the combination and assessed for tumor growth inhibition. Results: The combination of adavosertib and navitoclax resulted in greater antiproliferative effects in vitro compared to either single agent (p< 0.05). This effect was classified as synergistic with CI values Conclusions: The combination of adavosertib and navitoclax is active in preclinical TNBC models and induces apoptosis and tumor growth inhibition. This data supports the continued development of this combination in TNBC with investigation of potential selective markers. Citation Format: Simmons DM, Tse TE, Dailey K, Hartman SJ, Bagsby S, Pitts TM, Tentler JJ, Diamond JR. Rational combination of Wee1 and BCL-2 inhibition in preclinical models of triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-15.

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-Rag2nullIl2rγnullSIRPαNOD (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.

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 non-toxic 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 versus 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 post-treatment termination. However, combination-regimen displayed enhanced and sustained efficacy. Mechanistic assessments revealed that overcoming GEM resistance by co- administration with BMJ was possibly due to modulation of GEM transport/ metabolism pathway molecules [Ribonucleotide reductase regulatory subunit M1 (RRM1), human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (dCK)]. 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. Additionally, given their specific effectiveness against KRAS- mutant tumors, this combination could be potentially beneficial to a broader PanC patient population.

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.

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&reg, 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 &lt, 0.05 combo vs. adavosertib or capecitabine, TNBC013, p &lt, 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 &gamma, H2AX 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.

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.

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.

Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype defined by the lack of expression of the estrogen and progesterone receptors and lack of HER2 over-expression. ENMD-2076 is an orally bioavailable small molecule inhibitor of Aurora and angiogenic kinases with pro-apoptotic and antiproliferative activity in preclinical models of TNBC. Methods: This two institution, single-arm, two-stage, phase II clinical trial enrolled patients with locally advanced or metastatic TNBC refractory to 1-3 prior lines of chemotherapy in the advanced setting. Patients had ECOG PS ≤ 1, measureable disease by RECIST 1.1 and no evidence of brain metastasis. Patients were treated with ENMD-2076 250 mg PO daily with continuous dosing in 4-week cycles until disease progression or unacceptable toxicity occurred. The primary end point was 6-month clinical benefit rate (6-CBR) and secondary endpoints included time to progression (TTP), PK profile, safety and biologic correlatives in archival and fresh serial tumor biopsies in a subset of patients. Results: Between July 2012 and October 2016, 41 patients were enrolled (median age 54; range 30-73; female 40; male 1). Patients received a mean 1.7 prior lines of chemotherapy for locally advanced unresectable or metastatic disease and 80.5% received prior neoadjuvant or adjuvant chemotherapy (N=33). Thirty-six patients were evaluable per protocol for the primary efficacy analysis. Five patients (12.2%) were not included in the efficacy analysis due to: adverse events (AE) leading to discontinuation prior to objective efficacy assessment (N=3), not meeting eligibility criteria on day 1 (N=1) and withdraw of consent in cycle 1 (N=1). The study proceeded to the second stage of enrollment based on observing three 6-CBR events in Stage 1 (N=18 patients). The 6-CBR in the overall trial was 16.7% (95% exact CI: 6%-32.8%; 2 patients with PR and 4 patients with SD > 6 mos). The median duration of response or clinical benefit in these patients was 32 weeks (8 cycles). 4-CBR was 27.8% (95% exact CI: 14%-45.2%). Dose reduction occurred in 8 patients (20%) for fatigue, hypertension and proteinuria. The most common grade 3 treatment-related adverse events were hypertension (37.5%) and fatigue (10%). One patient experienced grade 4 hypertension. Analysis of serial tumor biopsies prior to and following 2 weeks of ENMD-2076 (N=8 patients), demonstrated a treatment-induced decrease in cellular proliferation (Ki-67) and microvessel density (CD34) as assessed by IHC. Immunofluorescence performed on a subset of samples demonstrated an increase in p53-family member expression following treatment, consistent with changes observed in preclinical TNBC patient-derived tumor xenograft models. Conclusions: ENMD-2076 has durable clinical activity in a subset of patients with pretreated, advanced or metastatic triple-negative breast cancer. Predictive biomarker development using archival and fresh tumor tissue is underway. Exploration of lower doses of ENMD-2076 in future clinical trials may improve tolerability. Citation Format: Diamond JR, Eckhardt SG, Pitts TM, van Bokhoven A, Aisner D, Gustafson DL, Capasso A, Elias AD, Storniolo AM, Schneider BP, Gao D, Tentler JJ, Borges VF, Miller KD. Clinical safety and efficacy of the aurora and angiogenic kinase inhibitor ENMD-2076 in previously treated, locally advanced or metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-16.

Background: RX-5902 (Supinoxin) is a novel anti-cancer compound that targets phosphorylated p68 RNA helicase, a member of the DEAD box family of helicases, affecting upstream and downstream molecules in the Wnt canonical pathway. As a single agent, RX-5902 exhibits strong growth inhibition in both in vitro and in vivo settings. Specifically, RX-5902 enhances survival and tumor growth inhibition in numerous xenograft models, including ovarian, renal and breast. We have previously shown RX-5902 inhibits cell growth in a dose-dependent fashion in the triple-negative breast cancer (TNBC) xenograft MDA-MB231. In the current study, we have expanded our investigation of the therapeutic potential of RX-5902 against TNBC using both in vitro and in vivo preclinical models. Methods: RX-5902 was provided by Rexahn, Inc. (Rockville, MD). Cell proliferation was measured using the Cell-Titer Glo luminescent cell viability assay (Promega). Apoptosis was assessed using Incucyte Caspase 3/7 Green apoptosis assay (Essenbioscience). Immunoblots of MDA-MB-231 cell line were probed for ß-catenin (Cell Signaling). Syngeneic 4T1 murine TNBC mice were obtained from Sippr-BK Laboratory Animal Co (Shanghai, China) and tumor volumes were measured twice a week. When the mean tumor volumes reached ˜90 mm3, mice were randomized and treated with vehicle or RX-5902 PO daily alone or in combination with anti-CTLA4 or anti-PD-1 BIW for 3 weeks. Tumor growth inhibition (TGI) was calculated at Day 25. Results: A panel of 18 TNBC cell lines were treated with RX-5902 and effects on cell proliferation were measured by the Cell Titer-Glo assay. Using 100nM as a cutoff, 14 sensitive lines and 4 resistant lines were identified, with an average IC50 of 56 nM in the sensitive lines. Of these, we chose 2 sensitive lines (MDA-MB-231, HCC1806) and 2 resistant lines (MDA-MB-436 and CAL-120) and assessed induction of apoptosis by the Incucyte caspase activity assay. Robust induction of apoptosis was observed in both sensitive lines (N=3). These lines were then subjected to cell cycle analysis by flow cytometry, which revealed a pronounced G2/M cell cycle arrest and aneuploidy following exposure to RX-5902. Western blot analysis of the MDA-MB-231 cell line showed decreases in the Wnt pathway-related protein nuclear ß-catenin in doses ranging from 20 nM to 200 nM. Finally, the therapeutic efficacy of RX-5902 was assessed as a single agent and in combination with two immune-oncology agents in the treatment of the TNBC 4T1 animal model. RX-5902 as a single agent showed dose dependency in the 4T1 model, and when given in combination with either anti-CTLA4 or anti-PD1 showed an additive effect (p Conclusions: RX-5902 showed efficacy against several in vitro and in vivo preclinical models of TNBC. RX-5902 resulted in G2/M arrest and induced apoptosis in sensitive TNBC cell lines and decreases in nuclear beta-catenin. In vivo, RX-5902 demonstrated additive anti-tumor effects when combined with either anti-CTLA4 or anti-PD1 immunotherapies. Together, these finding indicate that RX-5902 may have important clinical implications for the treatment of TNBC. A phase 2a clinical study in metastatic TNBC is ongoing..training_cert Citation Format: Tentler JJ, Frank JG, Kim DJ, George C, Lee YB, Ely B, Tan AC, Kim J, Pitts TM, Capasso A, Dailey KL, Eckhardt G, Diamond JR. Preclinical studies of RX-5902, a beta-catenin modulator in triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-16.

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 (IC50) 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 IC50 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

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 (ALKATI) was reported in 11% of melanomas but the response of melanomas expressing ALKATI 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 ALKATI 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 ALKATI were not sensitive to ALK inhibitors. In addition, a patient with mucosal melanoma expressing ALKATI 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 ALKATI 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 ALKATI. Mol Cancer Ther; 17(1); 222–31. ©2017 AACR.

// Justin Greene 1, * , Anna Nguyen 1, * , Stacey M. Bagby 1 , Gemma N. Jones 4 , WM. Tai 1, 3 , Kevin S. Quackenbush 1 , Anna Schreiber 1 , Wells A. Messersmith 1 , Kalpana M. Devaraj 2 , Patrick Blatchford 1 , S. Gail Eckhardt 1 , Elaine B. Cadogan 4 , Gareth D. Hughes 4 , Aaron Smith 4 , Todd M. Pitts 1 and John J. Arcaroli 1 1 Division of Medical Oncology, University of Colorado Anschutz Medical Campus and University of Colorado Cancer Center, Aurora, CO, USA 2 Pathology Department, University of Colorado Anschutz Medical Campus, Aurora, CO, USA 3 Division of Medical Oncology, National Cancer Centre Singapore, Singapore 4 Innovative Medicines and Early Development, Oncology, AstraZeneca, Cambridge, UK * These authors contributed equally to this work Correspondence to: John J. Arcaroli, email: john.arcaroli@ucdenver.edu Keywords: ATM; DNA damage; IRN; PDTX; CRC Received: August 06, 2017 Accepted: November 09, 2017 Published: December 05, 2017 ABSTRACT 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.

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 ( 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.

Ephrin B2 is variably expressed on tumor cells and its blockade has been shown to inhibit angiogenesis in animal models of pancreatic, colorectal, lung and head, and neck squamous cell carcinomas. However, the implications of ephrinB2 expression in cancer patients have remained elusive. In this study, we analyzed the cancer genome atlas (TCGA) for ephrinB2 expression. We report significant correlations between EFNB2 expression, overall survival and disease-free survival in head and neck squamous cell carcinoma (HNSCC, n = 519), pancreatic adenocarcinoma (n = 186), and bladder urothelial carcinoma (n = 410). In HNSCC patients, high-EFNB2 mRNA expression was associated with tumor HPV negativity, oral cavity location, alcohol intake, higher TP53 mutation, and EGFR amplification. EphrinB2 overexpression also correlated with worse response to chemotherapy and radiotherapy. The therapeutic potential of blocking ephrinB2 was validated in HNSCC patient-derived tumor xenografts and showed significant improvement in survival and tumor growth delay. Our data shows that ephrinB2 overexpression can serve as a critical biomarker for patient prognosis and response to therapy. These results should guide design of future clinical trials exploring EphrinB2 inhibition in cancer patients. © 2016 Wiley Periodicals, Inc.

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 (IC50

// Todd M. Pitts 1, 3, * , Erica L. Bradshaw-Pierce 2, 3, 5, * , Stacey M. Bagby 1 , Stephanie L. Hyatt 1 , Heather M. Selby 1 , Anna Spreafico 1 , John J. Tentler 1, 3 , Kelly McPhillips 1 , Peter J. Klauck 1 , Anna Capasso 1 , Jennifer R. Diamond 1, 3 , S. Lindsey Davis 1, 3 , Aik Choon Tan 1, 3 , John J. Arcaroli 1, 3 , Alicia Purkey 1 , Wells A. Messersmith 1, 3 , Jeffery A. Ecsedy 4 , S. Gail Eckhardt 1, 3 1 Division of Medical Oncology, School of Medicine, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA 2 Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA 3 University of Colorado Cancer Center, University of Colorado Anschutz Medical Campus, Aurora, CO, USA 4 Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited, Cambridge, MA, USA 5 Takeda California, San Diego, CA, USA * These authors have contributed equally to this work Correspondence to: Todd M. Pitts, email: Todd.Pitts@ucdenver.edu Keywords: colorectal cancer, aurora kinase a Received: January 20, 2016 Accepted: June 17, 2016 Published: July 1, 2016 ABSTRACT 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 IC 50 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 IC 50 s 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.

// Kevin S. Quackenbush 1 , Stacey Bagby 1 , Wai Meng Tai 1, 2 , Wells A. Messersmith 1 , Anna Schreiber 1 , Justin Greene 1 , Jihye Kim 1 , Guoliang Wang 1 , Alicia Purkey 1 , Todd M. Pitts 1 , Anna Nguyen 1 , Dexiang Gao 1 , Patrick Blatchford 1 , Anna Capasso 1 , Alwin G. Schuller 3 , S. Gail Eckhardt 1 , John J. Arcaroli 1 1 Division of Medical Oncology, University of Colorado Denver and University of Colorado Cancer Center, Denver, CO, USA 2 Division of Medical Oncology, National Cancer Centre Singapore, Singapore 3 AstraZeneca R & D Boston, Massachusetts, Waltham, Massachusetts, MA, USA Correspondence to: John J. Arcaroli, email: john.arcaroli@ucdenver.edu Keywords: CRC, WNT, IRN, NuMA, PDTX Received: December 30, 2015 Accepted: February 28, 2016 Published: April 07, 2016 ABSTRACT Background: Dysregulation of the canonical Wnt signaling pathway has been implicated in colorectal cancer (CRC) development as well as incipient stages of malignant transformation. In this study, we investigated the antitumor effects of AZ1366 (a novel tankyrase inhibitor) as a single agent and in combination with irinotecan in our patient derived CRC explant xenograft models. Results: Six out of 18 CRC explants displayed a significant growth reduction to AZ1366. There was one CRC explant (CRC040) that reached the threshold of sensitivity (TGII ≤ 20%) in this study. In addition, the combination of AZ1366 + irinotecan demonstrated efficacy in 4 out of 18 CRC explants. Treatment effects on the WNT pathway revealed that tankyrase inhibition was ineffective at reducing WNT dependent signaling. However, the anti-tumor effects observed in this study were likely a result of alternative tankyrase effects whereby tankyrase inhibition reduced NuMA levels. Materials and Methods: Eighteen CRC explants were treated with AZ1366 single agent or in combination for 28 days and treatment responses were assessed. Pharmacokinetic (AZ1366 drug concentrations) and pharmacodynamic effects (Axin2 levels) were investigated over 48 hours. Immunohistochemistry of nuclear β-catenin levels as well as western blot was employed to examine the treatment effects on the WNT pathway as well as NuMA. Conclusions: Combination AZ1366 and irinotecan achieved greater anti-tumor effects compared to monotherapy. Activity was limited to CRC explants that displayed irinotecan resistance and increased protein levels of tankyrase and NuMA.

Immunotherapies that target immune regulatory checkpoints such as CTLA-4 and PD-1 are widely used among many cancer types and have shown positive results in CRC with high microsatellite instability. However, in microsatellite stable (MSS) CRC there is a dismal response rate of 0%. The limited efficacy has shown to be partially due to the lack of T-cells in the tumor microenvironment and/or no activation/regulation of paramount cells in the immune system. The Wnt pathway is the most commonly altered pathway in CRC and is highly involved in driving tumor initiation and progression. Recent evidence also demonstrates the Wnt pathway is involved in T-lymphocyte development, maturation/activation of CD8+ effector T cells and recruitment of dendritic cells. Therefore, targeting the Wnt pathway utilizing a Porcupine (PORCN) inhibitor (ETC-159) in MSS CRC may be a promising strategy to sensitize tumors to immune checkpoint inhibition. Human Immune System BRGS (BALB/c, Rag2−/−, IL2RγC−/−, NODSIRPα) mice were engrafted with MSS CRC PDX (hPDX). The hPDX were randomized according to human chimerism into the following drug treatments groups: Vehicle, ETC-159, nivolumab, and the combination. Treatments began when tumors reached 100-300mm3 and tumors were measured twice weekly. At the end of study, sera, lymph nodes, spleen, and tumor tissue were collected for immunohistochemistry, single cell suspensions, and flow cytometry analysis. Combination therapy resulted in a significant decrease in tumor volume compared to both single agents and vehicle. Flow cytometric analysis demonstrated an increase in human immune cells, in particular human CD4 and CD8 cells in the combination compared to the vehicle and nivolumab treated groups. Additionally, these T-cells showed increased signs of activation and effector function, as indicated by increased CD69+ expression, effector memory subsets, and granzyme B+ cells in the TILs, with a further reduction in Treg populations, suggesting an overall increase in inflammation. An increase in MHC II expression on tumor cells was observed in the ETC-159 single agent with a statistically significant increase in the combination treated tumors demonstrating enhanced antigen presentation. Furthermore, PD-1 expression was upregulated on CD4+ T-cells in the ETC-159 single agent. Lastly, VECTRA analysis corroborates the flow cytometry data showing a changing tumor immune landscape through an increase in CD4+ and CD8+ T cells in the tumor and surrounding stroma. Our data demonstrates the combination treatment of ETC-159 + nivolumab in MSS CRC hPDX show increased tumor infiltration of human immune cells. Further preclinical data is compulsory but these results support further development of this combination in clinical trials. Citation Format: Stacey M. Bagby, Sarah J. Hartman, Natalie M. Navarro, Betelehem W. Yacob, Jeremy Shulman, Jessica Barkow, Christopher H. Lieu, S. Lindsey Davis, Alexis D. Leal, Wells A. Messersmith, Angela Minic, Kimberly R. Jordan, Julie Lang, Todd M. Pitts. Sensitizing microsatellite stable colorectal cancer to immune checkpoint therapy utilizing Wnt pathway inhibition [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 6647.

Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer deaths with more than 56,000 new cases estimated to be diagnosed in 2019. Current treatment options for PDAC include radiation and chemotherapeutic regimens, however these targeted therapies are ineffective for patients with advanced disease progression. Additionally, the dense stromal nature of PDAC tumors create challenges to target the cancer cells resulting in incomplete cell killing and eventual drug resistance. Recent evidence has shown that CPT1A, an enzyme that regulates the entry of lipids into mitochondria for β-oxidation, is strongly expressed in several cancers. CPT1A is located on the mitochondrial membrane and potentially interacts with BCL-2, an anti-apoptotic protein that promotes tumor maintenance and metastasis. Metabolic stress can activate the anti-apoptotic effects of BCL-2, reprograming metabolism to use fat oxidation for cancer survival. Therefore, a co-inhibition using the selective BCL-2 inhibitor, venetoclax, with agents that inhibit CPT1A and β-oxidation, could be a novel strategy for PDAC. There are few studies considering CPT1A as a therapeutic target for PDAC. Current available drugs to target these pathways include the anti-anginal ranolazine, and CPT1A inhibitors etomoxir and perhexiline. Previous studies have shown that expression of BCL-2 by tumor cells is necessary for BCL-2 inhibitors to be effective. We initially wanted to determine the expression of BCL-2 and CPT1A in PDAC cells utilizing western blot and rtPCR, and to confirm their proximity using a proximity ligation assay (PLA). PDAC cells were then plated in 96 well plates and Cell Titer-Glo assays were performed to determine effective concentrations of single agent venetoclax, etomoxir, and perhexiline. The effects of these drugs in combination were then evaluated using a clonogenic assay, which was analyzed using the ImageJ colony area plugin. PDAC cells were then exposed to the combinations and western blots were performed to evaluate changes downstream effectors. We have confirmed the expression of BCL-2 and CPT1A on the mitochondrial membrane using Westerns, rtPCR, and a PLA on several PDAC lines. Though single agent drugs had little effect on cell viability, the combination of venetoclax with CPT1A and β-oxidation inhibitors decreased colony formation in some PDAC cell lines. Western blot analysis revealed the drug combinations affected the phosphorylation of AKT and 4E-BP1 and expression of the pro-apoptotic protein BID. These data suggest that co-targeting BCL-2 and CPT1A have potential for anti-tumor effects in PDAC. Additional research into the role of CPT1A in PDAC biology will elucidate the optimal dosing concentrations and mechanisms for further studies. Citation Format: Sarah J. Hartman, Nathalie Nadales, Stacey M. Bagby, Betelehem W. Yacob, Brian L. Gittleman, Adriana Estrada-Bernal, Anh T. Le, Christopher H. Lieu, S. Lindsey Davis, Alexis D. Leal, Jennifer R. Diamond, Wells A. Messersmith, Isabel R. Schlaepfer, Todd M. Pitts. Therapeutic targeting of lipid oxidation and apoptosis in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6387.

4031 Background: The majority of pts with mCRC have microsatellite stable (MSS) tumors with minimal response to PD-L1/PD-1 blockade. MEK inhibition and VEGF inhibition have immunomodulatory effects (upregulation of tumor major histocompatibility complex-I expression, enhanced T-cell infiltration, reduced MDSCs and Tregs in tumors) supporting clinical evaluation of combined MEKi (B), anti–PD-1 (P), and anti-VEGF (BV) in pts with mCRC. We hypothesize that the combination of binimetinib, pembrolizumab, and bevacizumab (BPBV) will result in greater clinical benefit than pembrolizumab alone. Methods: Patients with chemotherapy-refractory mCRC were evaluated (20 planned in the safety lead-in and 50 planned for total accrual). B was dosed at 45mg PO BID, P was administered at 200mg IV Q21 days, and BV was administered at 7.5mg/kg IV Q21 days. Primary objectives were safety, tolerability, and investigator-assessed ORR by RECIST 1.1. Clinical benefit rate (CR+PR+SD) and progression-free survival were secondary endpoints. Descriptive statistics were used to summarize safety and clinical activity. Results: As of January 9, 2020, 21 pts (10 KRAS/NRASmt, 11 RASwt, 21 MSS) were enrolled into the safety lead-in and were evaluable. The median number of prior therapies was 6. The BPBV combination was tolerable. Treatment-related Gr 1-2 and Gr 3-4 AEs occurred at 60% and 38%, respectively. The most frequent related Gr 3-4 AEs were aceniform rash, diarrhea, and hypertension (19%, 14%, 14% respectively). No treatment-related Gr 5 AEs occurred. A total of 17 patients were evaluable for response. Confirmed PR was observed in 2 pts (12%). SD was noted in 14 patients (82%) leading to a clinical benefit rate of 94%. 1 patient had PD as the best response to treatment. Median PFS was 6.4 months (95% CI 4.2-8.9). Molecular determinants, immune biomarkers, and updated tumor assessments of response will be presented. Conclusions: B + P + BV demonstrated a tolerable safety profile and improvements in ORR and clinical benefit rate compared to those reported with SOC in heavily pretreated pts with mCRC. Objective responses observed in pts were durable, suggesting benefit of this novel combination in a patient population refractory to immune therapies. Clinical trial information: NCT03475004 .

Background: We have previously demonstrated the efficacy of pharmacologic targeting of Aurora Kinase A (AurA) in cell line and patient-derived xenograft (PDX) models of triple-negative breast cancer (TNBC). We observed that inherent or acquired resistance to AurA inhibitors was associated with a senescence phenotype and up-regulation of the PI3K/Akt/mTOR pathway. This provided the rationale to assess whether this resistance could be overcome with the combination of the AurA inhibitor alisertib with the TORC1/2 inhibitor TAK-228. In select TNBC PDX models, we found that there were highly significant anti-tumor effects of the combination compared to single agents. The purpose of the current study was to evaluate the potential role for alterations in tumor metabolic profiles in anti-tumor activity after dual inhibition of AurA and TORC1/2, as both pathways are known to mediate cellular metabolism and energy utilization in normal and cancer cells. Methods: Three TNBC PDX models were used for these studies. Tumor sections were implanted subcutaneously on bilateral flanks of athymic nude mice. When tumors reached ~150 mm3 mice were randomized into four treatment arms: vehicle; alisertib (30 mg/kg); TAK-228 (0.75 mg/kg); or the combination and dosed daily by oral gavage for 30 days (n=10 tumors per arm). At end of study, tumors were harvested and polar metabolites extracted for global ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS)-based metabolomics with a focus on central carbon and nitrogen metabolism. Metabolites were separated using a high throughput gradient-based C18 method and identified and validated manually against an in-house library of 800+ compounds. Results: Global metabolomics analysis of PDX tumors revealed a significant impact of TAK-228 treatment on fatty acid oxidation, resulting in increased levels of saturated fatty acids (C8, C10, C12) and palmitoleic acid, an increase in arginine, proline, metabolism and a decrease in glutamine metabolism. Alisertib treatment impacted tryptophan metabolism; initial results suggest a disruption of metabolite balance downstream of tryptophan. These disruptions in nitrogen and carbon metabolism occurred in the combination treatment arm as well. Of note, glucose utilization via glycolysis and the pentose phosphate pathway (PPP) were decreased with treatments, with the most profound decrease in the combination arm. Conclusions: In addition to increased anti-tumor growth activity, TNBC PDX tumor metabolic profiles were altered in mice treated alisertib, TAK-228 and the combination. Global metabolomics analysis revealed that several key metabolic pathways, including fatty acid oxidation, amino acid metabolism, glycolysis and PPP activity were significantly dysregulated, likely contributing to the observed tumor growth inhibition. Importantly, the effects on several pathways by the single agents persisted in the combination, while others, including the decrease in glycolysis and PPP activity, were more pronounced in the combination arm. These results support the hypothesis that dual targeting of AurA and TORC1/2 may exert potent anti-tumor activity in TNBC through several mechanisms, including the disruption of tumor cell energetics. This combination is currently being evaluated in a Phase I clinical trial with planned expansion cohorts with serial tumor biopsies (NCT02719691). These clinical samples may provide a means for validation of these preclinical observations and allow continued elucidation of the potential antitumor metabolic reprogramming resulting from this dual targeting strategy and the clinical setting most likely to benefit from this combination. Citation Format: John J Tentler, Angelo D'Allesandro, Julie A Reisz, Kyrie L Dailey, Anna Capasso, Anastasia Ionkina, Stacey M Bagby, Todd M Pitts, Jennifer R Diamond. Combined aurora kinase A and mTORC 1/2 inhibition results in disruption of tumor cell energetics in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-04.

Purpose: A driving factor in pancreatic ductal adenocarcinoma (PDAC) treatment resistance is the tumor microenvironment, which is highly immunosuppressive. One potent immunologic adjuvant is radiotherapy. Radiation, however, has also been shown to induce immunosuppressive factors, which can contribute to tumor progression and formation of fibrotic tumor stroma. To capitalize on the immunogenic effects of radiation and obtain a durable tumor response, radiation must be rationally combined with targeted therapies to mitigate the influx of immunosuppressive cells and fibrosis. One such target is ephrinB2, which is overexpressed in PDAC and correlates negatively with prognosis. Experimental Design: On the basis of previous studies of ephrinB2 ligand-EphB4 receptor signaling, we hypothesized that inhibition of ephrinB2-EphB4 combined with radiation can regulate the microenvironment response postradiation, leading to increased tumor control in PDAC. This hypothesis was explored using both cell lines and in vivo human and mouse tumor models. Results: Our data show this treatment regimen significantly reduces regulatory T-cell, macrophage, and neutrophil infiltration and stromal fibrosis, enhances effector T-cell activation, and decreases tumor growth. Furthermore, our data show that depletion of regulatory T cells in combination with radiation reduces tumor growth and fibrosis. Conclusions: These are the first findings to suggest that in PDAC, ephrinB2–EphB4 interaction has a profibrotic, protumorigenic role, presenting a novel and promising therapeutic target.

Background Triple-negative breast cancer (TNBC) remains an aggressive breast cancer subtype with limited treatment options. ENMD-2076 is a small-molecule inhibitor of Aurora and angiogenic kinases with proapoptotic and antiproliferative activity in preclinical models of TNBC. Methods This dual-institution, single-arm, two-stage, phase II clinical trial enrolled patients with locally advanced or metastatic TNBC previously treated with one to three prior lines of chemotherapy in the advanced setting. Patients were treated with ENMD-2076 250 mg orally once daily with continuous dosing in 4-week cycles until disease progression or unacceptable toxicity occurred. The primary endpoint was 6-month clinical benefit rate (CBR), and secondary endpoints included progression-free survival, pharmacokinetic profile, safety, and biologic correlates in archival and fresh serial tumor biopsies in a subset of patients. Results Forty-one patients were enrolled. The 6-month CBR was 16.7% (95% CI, 6–32.8%) and included two partial responses. The 4-month CBR was 27.8% (95% CI, 14–45.2%), and the average duration of benefit was 6.5 cycles. Common adverse events included hypertension, fatigue, diarrhea, and nausea. Treatment with ENMD-2076 resulted in a decrease in cellular proliferation and microvessel density and an increase in p53 and p73 expression, consistent with preclinical observations. Conclusions Single-agent ENMD-2076 treatment resulted in partial response or clinical benefit lasting more than 6 months in 16.7% of patients with pretreated, advanced, or metastatic TNBC. These results support the development of predictive biomarkers using archival and fresh tumor tissue, as well as consideration of mechanism-based combination strategies. Trial registration ClinicalTrials.gov, NCT01639248. Registered on July 12, 2012.

MEK inhibition is of interest in cancer drug development, but clinical activity in metastatic colorectal cancer (mCRC) has been limited. Preclinical studies demonstrated Wnt pathway overexpression in KRAS-mutant cell lines resistant to the MEK inhibitor, selumetinib. The combination of selumetinib and cyclosporin A, a noncanonical Wnt pathway modulator, demonstrated antitumor activity in mCRC patient-derived xenografts. To translate these results, we conducted a NCI Cancer Therapy Evaluation Program–approved multicenter phase I/IB trial (NCT02188264) of the combination of selumetinib and cyclosporin A. Patients with advanced solid malignancies were treated with the combination of oral selumetinib and cyclosporin A in the dose escalation phase, followed by an expansion cohort of irinotecan and oxaliplatin-refractory mCRC. The expansion cohort utilized a single-agent selumetinib “run-in” to evaluate FZD2 biomarker upregulation and KRAS-WT and KRAS-MT stratification to identify any potential predictors of efficacy. Twenty and 19 patients were enrolled in dose escalation and expansion phases, respectively. The most common adverse events and grade 3/4 toxicities were rash, hypertension, and edema. Three dose-limiting toxicities (grade 3 hypertension, rash, and increased creatinine) were reported. The MTD was selumetinib 75 mg twice daily and cyclosporin A 2 mg/kg twice daily on a 28-day cycle. KRAS stratification did not identify any differences in response between KRAS-WT and KRAS-MT cancers. Two partial responses, 18 stable disease, and 10 progressive disease responses were observed. Combination selumetinib and cyclosporin A is well tolerated, with evidence of activity in mCRC. Future strategies for concept development include identifying better predictors of efficacy and improved Wnt pathway modulation. Significance: These findings translate preclinical studies combining selumetinib and cyclosporin into a phase I first-in-human clinical trial of such a combination in patients with advanced solid malignancies. Cancer Res; 78(18); 5398–407. ©2018 AACR.

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 inCTNNB1and secreted cortisol but not aldosterone. CU-ACC2 cells had aTP53mutation and loss ofMSH2consistent 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.

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.

Dysregulation of the Notch1 receptor has been shown to facilitate the development and progression of colorectal cancer (CRC) and has been identified as an independent predictor of disease progression and worse survival. Although mutations in the NOTCH1 receptor have not been described in CRC, we have previously discovered a NOTCH1 gene copy number gain in a portion of CRC tumor samples. Here, we demonstrated that a NOTCH1 gene copy number gain is significantly associated with worse survival and a high percentage of gene duplication in a cohort of patients with advanced CRC. In our CRC patient-derived tumor xenograft (PDTX) model, tumors harboring a NOTCH1 gain exhibited significant elevation of the Notch1 receptor, JAG1 ligand and cleaved Notch1 activity. In addition, a significant association was identified between a gain in NOTCH1 gene copy number and sensitivity to a Notch1-targeting antibody. These findings suggest that patients with metastatic CRC that harbor a gain in NOTCH1 gene copy number have worse survival and that targeting this patient population with a Notch1 antibody may yield improved outcomes.

Triple-negative breast cancer (TNBC) is an aggressive disease with a poor prognosis. Advances in the treatment of TNBC have been hampered by the lack of novel effective targeted therapies. The primary goal of this study was to evaluate the efficacy of targeting Aurora kinase A (AurA), a key regulator of mitosis, in TNBC models. A secondary objective was to determine the role of the p53 family of transcriptional regulators, commonly mutated in TNBC, in determining the phenotypic response to the AurA inhibitor alisertib (MLN8237). Alisertib exhibited potent antiproliferative and proapoptotic activity in a subset of TNBC models. The induction of apoptosis in response to alisertib exposure was dependent on p53 and p73 activity. In the absence of functional p53 or p73, there was a shift in the phenotypic response following alisertib exposure from apoptosis to cellular senescence. In addition, senescence was observed in patient-derived tumor xenografts with acquired resistance to alisertib treatment. AurA inhibitors are a promising class of novel therapeutics in TNBC. The role of p53 and p73 in mediating the phenotypic response to antimitotic agents in TNBC may be harnessed to develop an effective biomarker selection strategy in this difficult to target disease. Mol Cancer Ther; 14(5); 1117–29. ©2015 AACR.

Purpose Triple-negative breast cancer (TNBC) is a subtype associated with poor prognosis and for which there are limited therapeutic options. The purpose of this study was to evaluate the efficacy of ENMD-2076 in p53-mutated TNBC patient-derived xenograft (PDX) models and describe patterns of terminal cell fate in models demonstrating sensitivity, intrinsic resistance, and acquired resistance to ENMD-2076. Experimental design p53-mutated, TNBC PDX models were treated with ENMD-2076 and evaluated for mechanisms of sensitivity or resistance to treatment. Correlative tissue testing was performed on tumor tissue to assess for markers of proliferation, apoptosis, senescence, and pathways of resistance after treatment and at the time of acquired resistance. Results Sensitivity to ENMD-2076 200 mg/kg daily was associated with induction of apoptosis while models exhibiting intrinsic or acquired resistance to treatment presented with a senescent phenotype. Response to ENMD-2076 was accompanied by an increase in p53 and p73 levels, even within the background of mutant p53. Treatment with ENMD-2076 resulted in a decrease in pAurA and an increase in pHH3. We observed a TNBC subtype switch from the luminal androgen receptor to the basal-like subtype at acquired resistance. Conclusion ENMD-2076 has antitumor activity in preclinical models of p53-mutated TNBC. Increased levels of p53 and p73 correlated with sensitivity whereas senescence was associated with resistance to ENMD-2076. The novel finding of a TNBC subtype switch at time of acquired resistance may provide mechanistic insights into the biologic effects of selective pressure of anticancer treatments on TNBC. ENMD-2076 is currently being evaluated in a Phase 2 clinical trial in patients with metastatic, previously treated TNBC where these biologic correlates can be further explored.

Background Dysregulation of the Src pathway has been shown to be important at various stages of cancer. Dasatinib is a potent Src/Abl inhibitor and has demonstrated to have anti-proliferative and anti-invasive activity in many preclinical models. The objective of this study was to determine the anti-tumor activity of dasatinib using in vitro and in vivo preclinical colorectal (CRC) models. Methods CRC cell lines and patient-derived tumor explant (PDX) models were used to investigate the efficacy of dasatinib. We treated 50 CRC cell lines with dasatinib for 72 hours and proliferation was assayed by a sulforhodamine B (SRB) assay; an IC50 ≤ 0.08 μmol/L was considered sensitive. We treated 17 patient-derived CRC explants with dasatinib (50 mg/kg/day, administered once-daily) for 28 days to determine in vivo efficacy. Tumor growth inhibition (TGI) ≥ 50% was considered sensitive. Results We found that 8 out of 50 CRC cell lines reached an IC50 ≤ 0.08 μmol/L with dasatinib treatment. In addition, of 17 CRC explants grown in the xenograft mouse model, 2 showed sensitivity to dasatinib. The anti-tumor effects observed in this study were a result of G1 cell cycle arrest as the dasatinib sensitive CRC cell lines exhibited G1 inhibition. Moreover, those CRC cell lines that were responsive (0.08 μmol/L) to treatment demonstrated a significant baseline increase in Src and FAK gene expression. Conclusion Dasatinib demonstrated significant anti-proliferative activity in a subset of CRC cell lines in vitro, especially in those with increased Src expression at baseline, but only showed modest efficacy in CRC explants. Dasatinib is currently being studied in combination with chemotherapy in patients with advanced CRC, as its use as a single agent appears limited.

Antiangiogenic therapy is commonly used for the treatment of colorectal cancer (CRC). Although patients derive some clinical benefit, treatment resistance inevitably occurs. The MET signaling pathway has been proposed to be a major contributor of resistance to antiangiogenic therapy. MET is upregulated in response to vascular endothelial growth factor pathway inhibition and plays an essential role in tumorigenesis and progression of tumors. In this study, we set out to determine the efficacy of cabozantinib in a preclinical CRC patient-derived tumor xenograft model. We demonstrate potent inhibitory effects on tumor growth in 80% of tumors treated. The greatest antitumor effects were observed in tumors that possess a mutation in the PIK3CA gene. The underlying antitumor mechanisms of cabozantinib consisted of inhibition of angiogenesis and Akt activation and significantly decreased expression of genes involved in the PI3K pathway. These findings support further evaluation of cabozantinib in patients with CRC. PIK3CA mutation as a predictive biomarker of sensitivity is intriguing and warrants further elucidation. A clinical trial of cabozantinib in refractory metastatic CRC is being activated.

Background: AZD0156 is an oral inhibitor of ATM, a serine threonine kinase that plays a key role in DNA damage response associated with DNA double strand breaks. Topoisomerase-I inhibitors like irinotecan induce single-strand DNA breaks, which are converted to double-strand breaks during DNA replication. Thus the combination of AZD0156 and irinotecan is a rational combination for clinical use. Irinotecan is used clinically to treat a variety of malignancies, including colorectal cancer (CRC), usually in combination with 5-fluorouracil (5FU) as FOLFIRI. An ongoing phase 1 clinical trial is evaluating AZD0156 in combination with single-agent irinotecan and FOLFIRI in patients with refractory cancers (NCT02588105). The purpose of this study is to evaluate AZD0156 in combination with irinotecan and 5FU in preclinical models of CRC to help inform clinical use. Methods: Anti-proliferative effects of single-agent AZD0156 and combination therapy with SN38 (active metabolite of irinotecan) and 5FU were evaluated in CRC cell lines using the Cell-Titer Glo assay. Immunoblotting and cell cycle analysis were performed to determine the mechanism of enhanced combination effects. Four CRC patient derived xenograft (PDX) models were treated with AZD0156, irinotecan, and 5FU alone and in combination for assessment of tumor growth inhibition (TGI). Results: An enhanced antiproliferative effect was observed with the combination treatment over either single agent. A more significant synergistic effect was demonstrated with the combination of AZD0156 and SN38 as compared with the combination of AZD0156 and 5FU. Cell cycle data demonstrated enhanced cell cycle arrest with combination therapy as compared to single agents. Immunoblotting results suggest a decrease in phosphorylated gamma-H2AX in cell lines treated with combination therapies. Increased TGI was observed in CRC PDX models treated with the combination of AZD0156 and irinotecan as compared to single-agent therapy in 3 of 4 models. There was not a significant change in TGI with the addition of 5FU for triplet therapy in the majority of models. Conclusions: The combination of AZD0156 with irinotecan is synergistic in in vitro models and is associated with increased TGI in CRC PDX in vivo models. The addition of 5FU to AZD0156 and irinotecan did not result in increased TGI as compared to doublet therapy in CRC PDX models, though did not decrease the AZD0156/irinotecan combination effect. An ongoing clinical trial is evaluating this combination in patients with cancers refractory to standard treatments (NCT02588105). Citation Format: S. Lindsey Davis, Marina I. Schlaepfer, Stacey M. Bagby, Sarah J. Hartman, Betelehem W. Yacob, Tonia Tse, Dennis M. Simmons, Jennifer R. Diamond, Christopher H. Lieu, Alexis D. Leal, Elaine B. Cadogan, Gareth D. Hughes, Stephen T. Durant, Wells A. Messersmith, Todd M. Pitts. Ataxia telangiectasia mutated (ATM) kinase inhibitor AZD0156 in combination with 5-fluorouracil and irinotecan in preclinical models of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4720.

Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype defined by the lack of expression of the estrogen and progesterone receptors and lack of HER2 over-expression. TNBC carries an increased risk of developing distant metastatic disease and cancer-related death. TNBC is heterogeneous in its underlying biology, however, mutations in p53 are found in approximately 80% of tumors. The purpose of this study was to utilize TNBC cell line models to investigate the role of p53 and p73 in mediating sensitivity to MLN8237, a selective Aurora kinase A inhibitor, in the setting of mutant or wildtype p53. Additionally, we used p53 and p73 knock-down models to determine the role of p53 and p73 in mediating induction of apoptosis and cellular senescence in response to Aurora kinase inhibition. Methods: Eighteen TNBC cell lines were exposed to MLN8237 and the effects on proliferation, apoptosis, and cell cycle distribution were evaluated. Proliferation was assessed using an SRB assay, apoptosis was analyzed using a caspase 3/7 assay and cell cycle was measured using flow cytometry. Proliferation was confirmed using a cell counting technique to control for an increase in cell size following MLN8237 exposure. The p53 wildtype TNBC cell line CAL51 was transduced with several clones of shRNA constructs targeting p53 and p73 with > 80% knockdown by RT-PCR. These clones were exposed to escalating doses of MLN8237 and the effect on proliferation was determined using an SRB assay and the effect on cellular senescence was determined using a senescence associated beta-galactosidase (SA b-gal) assay. Results: In vitro exposure to MLN8237 resulted in robust inhibition of proliferation in TNBC cell lines which was associated with dose-dependent G2/M cell cycle arrest and induction of caspase-dependent apoptosis in a subset of sensitive cell lines. Knock-down of p53 and p73 in the CAL51 cell line resulted in an increase in the absolute 50% inhibitory concentration (IC50) calculated from the SRB proliferation assay curves from 0.04 μM to > 2 μM, 1.8 μM, > 2 μM and 1.5 μM in the CAL51scr, CAL51p5310, CAL51p5312, CAL51p7326, CAL51p7355, respectively. The sensitive p53 mutated MDA-MB-468 cell line (IC50 40 nM) and the sensitive p53 wild-type CAL51 cell line (IC50 45 nM) were selected for further experimentation. Exposure to MLN8237 at concentrations of 50 nM and 100 nM for 7 days resulted in induction of cellular senescence as detected by the SA b-gal assay in the CAL51 p53 wild-type cell line, but not in the MDA-MB-468 cell line where induction of apoptosis at 48 hours was observed using the caspase 3/7 assay. In the CAL51 p73 knock-down clones, induction of cellular senescence was not observed following exposure to MLN8237. Conclusions: MLN8237 exhibited robust anticancer activity towards preclinical models of p53 mutated and p53 wild-type TNBC, supporting future clinical investigation of Aurora kinase inhibitors in TNBC. In p53 wild-type TNBC, both p53 and p73 mediate sensitivity to MLN8237 and p73 is essential for induction of cellular senescence following exposure to MLN8237. Biomarkers predictive of response to MLN8237 in p53 mutated TNBC are being developed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-06.

Background: Dysregulation of the Notch pathway has been identified to play an important role in the development and progression of colorectal cancer (CRC). In this study, we used a patient-derived CRC explant model to investigate the efficacy of the clinical γ-secretase inhibitor (GSI) PF-03084014. Methods: A total of 16 CRC explants were treated with PF-03084014. Knockdown of RBPjκ gene was used to determine the specificity of PF-03084014. Evaluation of the Notch and Wnt pathways in CRC explant tumours was performed by gene array and immunoblotting. Results: We identified a subset of CRC tumours that exhibited elevations of the Notch and Wnt pathways sensitive to PF-03084014. Treatment with the GSI resulted in a significant reduction in cleaved Notch, Axin2 (Wnt-dependent gene) and active β-catenin. In addition, knockdown of the RBPjκ gene showed that PF-03084014 has specificity for the Notch pathway in an HCT116 cell line xenograft model. Finally, an increase in apoptosis was observed in CRC001- and CRC021-sensitive tumours. Conclusion: This study provides evidence that inhibition of γ-secretase may be beneficial in a subset of patients with elevated levels of the Wnt and Notch pathways.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Purpose: Dysregulation of the phosphoinositide 3-kinase (PI3K) and Src signaling pathways commonly occur in colorectal cancer. Mutations in the PIK3CA gene are associated with an increase in severity of disease and worse clinical outcomes. Elevated levels of Src have been identified in premalignant lesions and are suggested to play a central role in tumor progression. Because these pathways appear to enhance tumor growth and metastasis, molecularly targeted agents for both pathways are currently being evaluated in early-phase clinical trials. Experimental Design: We used colorectal cancer cell lines and a patient-derived explant model to investigate the efficacy of saracatinib. Mutations in the PIK3CA were evaluated to examine the association between mutations in the PIK3CA gene and sensitivity to saracatinib. Results: We have identified a subset of patients with a PIK3CA (exon 9 and 20) mutation with increased sensitivity to saracatinib. A novel 3′ untranslated region (UTR) mutation was also shown to be associated with increased sensitivity to saracatinib and have a reduced affinity for miR-520a and miR-525a. Importantly, we show that Src inhibition reduces the interaction between Src and p85, subsequently decreasing Akt-dependent signaling. Conclusion: These results indicate that a personalized approach in targeting Src in PIK3CA-mutant patients with colorectal cancers may prove effective in a subset of patients with this genetic alteration. Clin Cancer Res; 18(9); 2704–14. ©2012 AACR.

Progress in oncology drug development has been hampered by a lack of preclinical models that reliably predict clinical activity of novel compounds in cancer patients. In an effort to address these shortcomings, there has been a recent increase in the use of patient-derived tumour xenografts (PDTX) engrafted into immune-compromised rodents such as athymic nude or NOD/SCID mice for preclinical modelling. Numerous tumour-specific PDTX models have been established and, importantly, they are biologically stable when passaged in mice in terms of global gene-expression patterns, mutational status, metastatic potential, drug responsiveness and tumour architecture. These characteristics might provide significant improvements over standard cell-line xenograft models. This Review will discuss specific PDTX disease examples illustrating an overview of the opportunities and limitations of these models in cancer drug development, and describe concepts regarding predictive biomarker development and future applications.

Purpose: ENMD-2076 is a unique orally bioavailable Aurora kinase and VEGFR inhibitor. The purpose of this phase 1 study of ENMD-2076 was to determine the MTD, pharmacokinetic, and pharmacodynamic profiles and preliminary antitumor activity. Experimental Design: Patients with refractory advanced solid malignancies were treated with ENMD-2076 orally with continuous once daily dosing. Doses from 60 to 200 mg/m2 were evaluated using a standard 3 (to 4) + 3 design. Pharmacokinetic parameters were studied on days 1, 28, and 30 to 35 of cycle 1. Expanded MTD cohorts included patients with ovarian cancer, colorectal cancer, and refractory solid tumors. Results: A total of 67 patients (46 F, 21M; ages 30–76) entered the study. Dose levels of 60, 80, 120, 200, and 160 mg/m2 were evaluated. Two patients experienced grade 3 hypertension at 200 mg/m2, and additional grade 3 neutropenia events limited tolerability at this dose. An intermediate dose of 160 mg/m2 was determined to be the MTD. The most common drug-related adverse events included hypertension, nausea/vomiting, and fatigue. The pharmacokinetics of ENMD-2076 were characterized by a rapid absorption phase (Tmax 3–7.8 hours), a t1/2 of 27.3 to 38.3 hours after a single dose, and dose proportional exposure. Decreased plasma sVEGFR2 was observed posttreatment. Two patients with platinum refractory/resistant ovarian cancer had RECIST partial responses. Conclusions: ENMD-2076 was well tolerated, had a linear pharmacokinetic profile, and showed promising antitumor activity, particularly in ovarian cancer. The recommended phase 2 dose of ENMD-2076 is 160 mg/m2 administered orally once daily with continuous dosing. Clin Cancer Res; 17(4); 849–60. ©2010 AACR.

Mutant K-ras activity leads to the activation of the RAS/RAF/MEK/ERK pathway in approximately 44% of colorectal cancer (CRC) tumors. Accordingly, several inhibitors of the MEK pathway are under clinical evaluation in several malignancies including CRC. The aim of this study was to develop and characterize predictive biomarkers of response to the MEK1/2 inhibitor AZD6244 in CRC in order to maximize the clinical utility of this agent. Twenty-seven human CRC cell lines were exposed to AZD6244 and classified according to the IC50 value as sensitive (≤0.1 μmol/L) or resistant (>1 μmol/L). All cell lines were subjected to immunoblotting for effector proteins, K-ras/BRAF mutation status, and baseline gene array analysis. Further testing was done in cell line xenografts and K-ras mutant CRC human explants models to develop a predictive genomic classifier for AZD6244. The most sensitive and resistant cell lines were subjected to differential gene array and pathway analyses. Members of the Wnt signaling pathway were highly overexpressed in cell lines resistant to AZD6244 and seem to be functionally involved in mediating resistance by shRNA knockdown studies. Baseline gene array data from CRC cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which predicted with 71% accuracy which of a test set of patient-derived K-ras mutant CRC explants would respond to AZD6244, providing the basis for a patient-selective clinical trial. These results also indicate that resistance to AZD6244 may be mediated, in part, by the upregulation of the Wnt pathway, suggesting potential rational combination partners for AZD6244 in CRC. Mol Cancer Ther; 9(12); 3351–62. ©2010 AACR.

Purpose: There is growing evidence implicating the importance of the insulin-like growth factor (IGF) pathway in colorectal cancer based upon the results of population studies and preclinical experiments. However, the combination of an IGF-I receptor (IGF-IR) inhibitor with standard colorectal cancer chemotherapies has not yet been evaluated. In this study, we investigated the interaction between PQIP, the dual IGF-IR/insulin receptor tyrosine kinase inhibitor, and standard chemotherapies in colorectal cancer cell line models. Experimental Design: The antiproliferative effects of PQIP, as a single agent and in combination with 5-fluorouracil, oxaliplatin, or SN38, were analyzed against four colorectal cancer cell lines. Downstream effector proteins, apoptosis, and cell cycle were also assessed in the combination of PQIP and SN-38. Lastly, the efficacy of OSI-906 (a derivative of PQIP) combined with irinotecan was further tested using a human colorectal cancer xenograft model. Results: Treatment with the combination of PQIP and each of three chemotherapies resulted in an enhanced decrease in proliferation of all four colorectal cancer cell lines compared with single-agent treatment. This inhibition was not associated with a significant induction of apoptosis, but was accompanied by cell cycle arrest and changes in phosphorylation of Akt. Interestingly, antitumor activity between PQIP and SN-38 in vitro was also reflected in the human colorectal cancer xenograft model. Conclusions: Combination treatment with PQIP, the dual IGF-IR/insulin receptor tyrosine kinase inhibitor, and standard colorectal cancer chemotherapy resulted in enhanced antiproliferative effects against colorectal cancer cell line models, providing a scientific rationale for the testing of OSI-906 and standard colorectal cancer treatment regimens. Clin Cancer Res; 16(22); 5436–46. ©2010 AACR.

Lessons Learned The maximum tolerated dose of the combination of linsitinib and irinotecan is linsitinib 450 mg daily on days 1–3 every 7 days and irinotecan 125 mg/m2 days 1 and 8 of a 21-day cycle. The adverse effects associated with the combination are not significantly increased beyond what is expected of each drug as a single agent. Multiple negative trials of insulin-like growth factor-1 receptor inhibitors performed in unselected patient populations led to the early discontinuation of linistinib development and this trial. Earlier integration of assessment of potential predictive biomarkers into clinical trials, as was planned in this study, is vital to the development of targeted therapies in oncology. Background This phase I dose-escalation study was designed to evaluate the safety and tolerability of the combination of irinotecan and insulin-like growth factor-1 receptor (IGF-1R) inhibitor linsitinib in patients with advanced cancer refractory to standard therapy. Methods Dose escalation in three specified dose levels was performed according to a standard 3 + 3 design. Dose levels were as follows: (a) linsitinib 400 mg and irinotecan 100 mg/m2, (b) linsitinib 450 mg and irinotecan 100 mg/m2, and (c) linsitinib 450 mg and irinotecan 125 mg/m2. Linisitinib was administered once daily on days 1–3, 8–10, and 15–17, and irinotecan on days 1 and 8. Assessment of a candidate predictive biomarker was planned in all patients, with further evaluation in an expansion cohort of advanced colorectal cancer. Results A total of 17 patients were treated, with 1 patient in both cohort 2 and 3 experiencing dose-limiting toxicity. Linsitinib 450 mg and irinotecan 125 mg/m2 was the maximum tolerated dose. Sixteen (94%) patients experienced at least one treatment-related adverse event. Neutropenia was the only grade >3 toxicity (4%). No significant hyperglycemia or QT interval prolongation was noted. No objective responses were observed; 47% (n = 8) had stable disease with median duration of 5.25 months. Conclusion Although the combination was determined safe, the study was halted due to termination of linsitinib development, and biomarker testing was not performed.

Pancreatic cancer (PanC) remains the 4th leading cause of cancer related-deaths in U.S. resulting in a dismal survival rate of Citation Format: Deepanshi Dhar, Dileep Kumar, Stacey Bagby, Todd M. Pitts, Wells A. Messersmith, Komal Raina, Rajesh Agarwal. Gaining insights into pancreatic cancer intervention with bitter melon, a natural agent, in combination with gemcitabine using patient derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 277.

Background: Colorectal cancer (CRC) ranks third in new cases and cancer deaths in the U.S. annually. The current front-line treatments for metastatic CRC (mCRC) are ineffective for an appreciable proportion of patients and induce significant toxicities. Thus, there is an urgent need for the development of new therapeutic strategies. Translation is a novel, underutilized target for cancer therapeutics. Eukaryotic elongation factor 2 (eEF2) is often overexpressed in CRC, causing upregulation of translation, upon which CRC may be dependent. Therefore, it may be possible to differentially target CRC cells by inhibiting translation. SVC112 is a novel inhibitor of translation and the parent molecule of SVC112 has been shown to lock eEF2 onto the ribosome. As such, this agent may be effective against mCRC by blocking translation of key oncogenes such as c-myc, which is overexpressed in mCRC. Methods: Forty-four CRC cell lines were exposed to SVC112 in vitro and CellTiter Glo ATP quantification was used to determine sensitivity or resistance based on IC50 values. Select sensitive and resistant cell lines were further validated with clonogenic assays to evaluate for sustained response. Immunoblotting was performed to assess levels of c-myc, survivin, and cyclin D1 in response to treatment with SVC112. Amino acid incorporation was measured using the Click-IT AHA kit protocol. eEF2 mRNA levels were ascertained from RNA seq data while cell cycle effects were determined by flow cytometry. Two cell line xenografts deemed to be sensitive based on IC50 values were treated in vivo with SVC112. Results: A subset of CRC cell lines were found to be sensitive to SVC112 in vitro, and notably, there was a trend towards increased eEF2 expression and greater responsiveness to SVC112. In sensitive cell lines, c-myc was downregulated by SVC112 in a dose-dependent manner whereas in resistant cell lines c-myc levels were not affected. Moreover, SVC112 caused a reduction of amino acid incorporation in sensitive cell lines compared to resistant cell lines. Likewise, the sensitive cell lines exhibited dose-dependent effects on the cell cycle with SVC112 whereas resistant cell lines did not. SVC112 demonstrates synergy with 5-FU in sensitive cell lines, while in the models and dosing conditions tested thus far, SVC112 has not demonstrated single agent efficacy in vivo. Conclusions: SVC112 demonstrates anticancer effects in a subset of CRC cell lines, indicating that translation machinery may be an effective druggable target in CRC. There is an apparent correlation between EF2 levels and sensitivity, which could be used as a selection strategy for sensitive tumors moving forward. SVC112’s ability to decrease amino acid incorporation and deplete c-myc in sensitive cell lines substantiates the proposed mechanism of action. Further experiments are needed to investigate the mechanism of resistance and whether this is related to anti-eEF2 exposure or a mechanism unrelated to eEF2 escape. Further analysis of SVC112 ribosomal engagement is ongoing. SVC112 has demonstrated synergistic effects with 5-FU, a standard chemotherapeutic for CRC, indicating its potential efficacy in combination therapy. Currently, PK analysis is under way and alternative dosing regimens in vivo may be warranted. Citation Format: Kelli M. Robertson, Annika L. Gustafson, John J. Tentler, Stacey M. Bagby, Capasso Anna, Peter J. Klauck, Todd M. Pitts, Jihye Kim, Aik Choon Tan, Tin Tin Su, S. Gail Eckhardt. Exploiting translation inhibition: antitumor efficacy of SVC112 in preclinical models of colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B051.

Background: Antibody-drug conjugates (ADCs) are an emerging technology consisting of antibody, linker, and toxic agent, which have the potential to offer a more targeted therapeutic approach than standard chemotherapy regimens. ADCs selectively bind to antigens expressed in tumor environments. An emerging target for the treatment of pancreatic cancer is guanylyl cyclase C (GCC). GCC is a transmembrane G protein receptor found on the apical surface of intestinal enterocytes. It plays an important role in GI homeostasis. GCC is highly expressed in various colorectal carcinomas as well as adenocarcinomas of the upper GI tract. The objective of this study was to determine the antitumorigenic activity of TAK-264, an investigational ADC that targets GCC. Experimental Procedures: The antiproliferative effects of TAK-264 were assessed in a panel of eleven pancreatic cancer cell lines with various molecular backgrounds. Additionally, ten unique pancreatic ductal adenocarcinoma (PDAC) cancer patient-derived xenograft (PDX) models were treated with TAK-264 and the efficacy was determined. Tumor size was evaluated twice per week by caliper measurements. Sensitivity to TAK-264 was defined based on tumor growth inhibition that was statistically significant when compared to the vehicle control. Baseline levels of GCC were analyzed by IHC/immunoblotting and RT-PCR 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 normal versus tumor tissue; a marked increase in GCC expression was observed in tumor tissue when compared to matching normal tissue. The in vitro experiments demonstrated a range of responses to TAK-264 in an SRB assay. Eight of the ten PDAC PDX models demonstrated a statistically significant tumor growth inhibition when compared to the vehicle control. Immunoblotting demonstrated an increase in phosphorylated-Histone-H3 in two cell lines and in the PDAC PDX models treated with TAK-264, indicating a DNA damage response. The analysis of GCC protein expression in normal versus tumor tissue has shown a marked increase in GCC expression in tumor tissue when compared to matching normal tissue. There was no correlation between baseline levels of GCC and response to TAK-264 in either PDX or cell line models. Conclusions: TAK-264, an ADC targeting GCC, has good growth suppression activity in pancreatic cancer cell lines and in pancreatic PDX models. These findings support a hypothesis that further investigation of ADC targeting GCC may lead to novel therapeutic modalities for pancreatic cancer. Citation Format: Anna R. Schreiber, Anna Nguyen, Stacey M. Bagby, Betelehem Yacob, Kevin Quackenbush, Joe L. Guy, Thomas Crowell, Bradley Stringer, Hadi Danaee, Thea Kalebic, Wells A. Messersmith, John J. Arcaroli, Todd M. Pitts. Evaluation of TAK-264, a novel antibody-drug conjugate in pancreatic cancer cell lines and patient-derived xenograft models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B050.

Introduction: Recent data have demonstrated that patients with MSI-H metastatic CRC are more likely to respond to a PD-1 inhibitor as a single agent compared to patients with MSS CRC. Effective immunotherapy approaches for MSS CRC remain a critical unmet need. There is great interest in investigating immune checkpoint inhibition in combination with novel agents, but preclinical studies have been hampered by current models. To gain a better understanding of immune responses and facilitate preclinical evaluation of combination strategies with immunotherapy, we developed a “hematopoietic” humanized mouse model with the intent of leveraging this model for the development of rational combinations in MSS CRC patients. Methods: BRGS (in house breeding) newborn pups were transplanted with CD34+ cells purified from (HLA unmatched) umbilical cord blood at 48 hours. In order to better understand the immune context of these novel animal models, we selected an MSI-high CRC (MDA-C0999-203) and a MSS (CRC172) humanized PDX model. At 16 weeks, MDA-C0999-203 (MSI-H) and CRC172 (MSS) were implanted on both flanks of humanized mice and nonhumanized controls. When the average tumor size reached ~150-300 mm3, both groups of mice were randomized into vehicle and nivolumab (30mg/kg IP twice weekly) stratified by % chimerism (human CD45+ >45%). At the end of the treatment, a portion of the tumor was immediately fixed in formalin for immunohistochemistry and pharmacodynamic analyses with seven-color multispectral imaging using the Perkin Elmer Vectra- 3 instrument, and compared to a surgical specimen from an untreated patient with an MSI-H tumor. Results: In the MSI-H model we observed tumor growth inhibition in the nivolumab-treated, humanized mice with respect to the humanized vehicle-treated control and the nonhumanized nivolumab-treated group (TGII 3.5% vs 76%). In a separate experiment, an MSS CRC humanized PDX model (CRC172) was treated with nivolumab as described above. Although initial control of tumor growth was observed, this was followed by rapid tumor progression (TGII 138.4%). In addition to flow cytometry, an MSI-H CRC tumor directly from a patient was stained for a variety of immunologic markers and analyzed by Vectra. The tumors contained both CD4- and CD8-positive cells, indicating T-cell infiltration. These data are consistent with what was observed by flow cytometry. Similar results were observed in an MSI-H CRC tumor grown as a xenograft in humanized mice both with and without nivolumab. However, in the MSS CRC PDX model, very few CD4 and CD8 cells were observed in the tumor or in the surrounding stroma. Conclusions: We have successfully established in vivo MSI-H and MSS humanized CRC PDXs. All mice were highly chimeric and the MSI-H model demonstrated high TILs, and responding tumors exhibited IFNγ production, high CD8%, higher effector memory % (HLADR+, CD45RO+), and decreased PD-L1 expression by flow cytometry. Interestingly, we also observed greater numbers of T cells in the lymph nodes of the MSI-H PDX. Moreover, immune infiltrates were observed in the MSI-H PDX compared to the MSS by immunohistochemistry. These data suggest that humanized PDX models may be useful in the development of rational combinations of immunotherapy. Citation Format: Anna Capasso, Julie Lang, Todd M. Pitts, Christopher H. Lieu, Scott Kopetz, Sarah L. Davis, Kimberly Jordan, Stacey M. Bagby, Wells A. Messersmith, Roberta Pelanda, S. Gail Eckhardt. Humanized patient-derived xenografts (PDXs) recapitulate clinical responses in microsatellite stable (MSS) and unstable (MSI-H) colorectal cancer (CRC) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A012.

Background: MLN0128 is an oral inhibitor of mTOR kinase and mTORC1/2 signaling. Alisertib is an oral inhibitor of Aurora A kinase. Senescence and upregulation of genes in the PI3K/AKT/mTor pathway have been observed in triple-negative breast cancer (TNBC) patient-derived xenograft models treated with alisertib, with greater tumor growth inhibition demonstrated in combination with MLN0128 as compared to each agent alone. An investigator-initiated trial was developed to evaluate the combination of MLN0128 and alisertib in patients with advanced solid tumors, followed by an expansion cohort in metastatic TNBC and other selected cancers. The goals of this ongoing study are to evaluate safety, tolerability, pharmacokinetics (PK) and preliminary efficacy of the combination. Results of dose escalation are presented here. Methods: Patients with advanced solid tumors refractory to standard therapy were treated orally at escalating doses with the combination of MLN0128 daily on a continuous schedule and alisertib twice daily (BID) on days 1-7 of a 21-day cycle. Dose escalation was conducted according to a standard 3+3 design. Key eligibility criteria included HgbA1c Citation Format: S. Lindsey Davis, Elaine T. Lam, Bradley R. Corr, Cindy L. O'Bryant, Ashley Glode, Nichole Adler, Todd M. Pitts, John J. Tentler, Anna Capasso, Kyrie Dailey, Natalie J. Serkova, Colin D. Weekes, Daniel L. Gustafson, Christopher H. Lieu, Wells A. Messersmith, Stephen Leong, S. Gail Eckhardt, Jennifer R. Diamond. A phase Ib study of the combination of MLN0128 (dual TORC1/2 inhibitor) and MLN8237 (Aurora A inhibitor, alisertib) in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A083.

Despite the availability of several Food and Drug Administration-approved drugs, advanced inoperable colorectal cancer remains incurable. In this study, we focused on the development of combined molecular targeted therapies against colon cancer by testing the efficacy of the combination of the histone deacetylase inhibitor vorinostat with the proteasome inhibitor bortezomib to determine if this resulted in synergistic antitumor effects against colorectal cancer. The effects of the histone deacetylase inhibitor vorinostat in combination with the proteasome inhibitor bortezomib on the growth of two colorectal cancer cell lines were assessed with regard to proliferation, cell cycle arrest, and apoptosis. Treatment with the combination of vorinostat and bortezomib resulted in a synergistic decrease in proliferation of both colorectal cancer cell lines compared with treatment with single agents alone. This inhibition was associated with a synergistic increase in apoptosis as measured by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase. In addition, we observed an increase in the proapoptotic protein BIM and in the number of cells arrested in the G2-M phase of the cell cycle. Although p21 levels were significantly increased, short hairpin RNA knockdown of p21 did not lead to changes in proliferation in response to the combination of drugs, indicating that although p21 is a target of these drugs, it is not required to mediate their antiproliferative effects. These data indicate that combination treatment with vorinostat and bortezomib result in synergistic antiproliferative and proapoptotic effects against colon cancer cell lines, providing a rational basis for the clinical use of this combination for the treatment of colorectal cancer. [Mol Cancer Ther 2009;8(2):342–9]

Background Vandetanib is a multitargeted tyrosine kinase inhibitor that affects vascular endothelial growth factor receptor (VEGF), epidermal growth factor (EGF), and rearranged during transfection (RET) mediated receptors which are important for growth and invasion of biliary and pancreatic cancers. This phase I study evaluated the safety profile of vandetanib in combination with standard doses of gemcitabine and capecitabine in order to determine the maximum tolerated dose (MTD). Methods In this single center phase I trial, patients received gemcitabine intravenously (IV) at 1000 mg/m2 days 1, 8, 15 in a 28 day cycle, capecitabine orally at 850 mg/m2 twice daily on days 1–21, and escalating doses of vandetanib (200 or 300 mg orally daily). Once the MTD was defined, an expansion cohort of patients with advanced biliary cancers and locally advanced or metastatic pancreatic cancer was enrolled. Blood samples were also collected at predetermined time points for biomarker analysis. Results Twenty-three patients were enrolled: 9 in the dose escalation and 14 in the dose expansion cohort. One dose limiting toxicity (DLT), of grade 4 neutropenia, occurred in the 200 mg vandetanib cohort. The most common adverse effects were diarrhea (39 %), nausea and vomiting (34 %), and rash (33 %). There were 3 partial responses and stable disease of >2 months (range 2–45, median 5) was observed in 15/23 patients. There was no association between changes in biomarker analytes and disease response. Conclusion The combination of gemcitabine, capecitabine and vandetanib is well tolerated at the recommended phase II dose of gemcitabine 1000 mg/m2 weekly for three consecutive weeks, capecitabine 850 mg/m2 BID days 1–21, and vandetanib 300 mg daily, every 28 days. This combination demonstrated promising activity in pancreaticobiliary cancers and further evaluation is warranted in these diseases. NCT00551096.

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.