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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.

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

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.

Genomic rearrangements resulting in activating kinase fusions have been increasingly described in a number of cancers including malignant melanoma, but their frequency in specific melanoma subtypes has not been reported. We used break-apart fluorescence in situ hybridization (FISH) to identify genomic rearrangements in tissues from 59 patients with various types of malignant melanoma including acral lentiginous, mucosal, superficial spreading, and nodular. We identified four genomic rearrangements involving the genes BRAF, RET, and ROS1. Of these, three were confirmed by Immunohistochemistry (IHC) or sequencing and one was found to be an ARMC10-BRAF fusion that has not been previously reported in melanoma. These fusions occurred in different subtypes of melanoma but all in tumors lacking known driver mutations. Our data suggest gene fusions are more common than previously thought and should be further explored particularly in melanomas lacking known driver mutations.

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.

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.

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.

Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling is activated in many cancers, and is associated with mutational loss of PTEN function, or activation of Phosphatidylinositide-3-kinase (PI3K). Activation of the PDPK1 signaling pathway contributes to proliferation and survival pathways in tumor cells. In a mouse Braf(V600E)::Pten(-/-) melanoma model genetic inactivation of PDKP1 delays the development of pigmented lesions and melanoma. We have found that the PDPK1 inhibitor PHT-427 has antitumor activity in BRAF(V600E)::NRAS(G12V) melanoma patient derived xenografts (PDX) grown in immune deficient mice. Pleckstrin homology (PH) domains are 100-120 amino acid domains found in more than 250 human proteins that bind phosphatidylinositide (Ptdlns) lipids in cell membranes. The phosphorylation of Ptdins, and the consequent binding of PH domain containing proteins is found in many signal transduction pathways critical for cell growth, survival, angiogenesis, and metastasis. Though the primary amino acid sequences of PH domains are not highly conserved, PH domains do have a highly conserved tertiary structure. This structural conservation of PH domains combined with the role PH domains play in signal transduction make them promising targets for small molecule inhibitors. PHT-427 was designed to bind PH domains, and experiments show that PHT-427 binds with the highest affinity to the PH domain of PDKP1. We wanted to test the antitumor activity of PHT-427 in human melanoma PDX models of varying genetic background. About 50% of melanoma tumors are driven by the BRAF(V600E) mutation, and these tumors are effectively targeted with BRAF inhibitors, such as vemurafenib. Unfortunately, these tumors usually develop resistance to vemurafenib, and the disease progresses. Additionally, 40-50% of melanoma tumors do not carry the BRAF(V600E) mutation, indicating the need to target additional pathways in melanoma. We sequenced human PDX samples on the Ion Proton sequencer, using the 409 gene Comprehensive Cancer Panel from Life Technologies. Compared to xenografts, PDXs are closer to the tumor biology of the patient, having a higher degree of molecular subtypes and intratumor heterogeneity, and a mixture of human and mouse stroma. The TMAP alignment program that works with Ion Torrent sequence data cannot handle a combined human-mouse reference genome, we developed a set of scripts we call Graft Extraction Out of REcipient Genome (GEORGE). Sequence reads aligned to the hg19 human reference, and mm10 mouse reference are evaluated by GEORGE and assigned to either the human, or mouse genome. In this project, only human assigned reads were used for variant calling. We sequence 14 melanoma samples, and five samples carried BRAF(V600E). We tested the antitumor activity of PHT-427 in four PDX samples with various BRAF and NRAS mutational statuses. We found that PHT-427 had antitumor activity in a vemurafenib resistant BRAF(V600E)::NRAS(G12V) mutant PDX sample. Citation Format: Brian P. James, Robert Lemos, Jr., Stacey Bagby, Steven Robinson, William Robinson, John Tentler, S. Gail Eckhardt, D. Lynn Kirkpatrick, Garth Powis. A PDK1 inhibitors that has antitumor activity in a vemurafenib resistant BRAF(V600E)::NRAS(G12V) melanoma PDX. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-29.

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.

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P

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.

Triple-negative breast cancer (TNBC) represents a challenge clinically due to a lack of response to hormonal and HER2-targeted agents coupled with an aggressive disease course. As the biology of this breast cancer subtype is better understood, it is clear that TNBC is a heterogeneous disease and one targeted therapy is unlikely to be active in all patients. Biomarkers predictive of response to treatment are thus of great importance in TNBC. This review outlines studies evaluating biomarkers predictive of response to neoadjuvant chemotherapy and to targeted therapies in the advanced setting. The development of validated biomarkers in conjunction with novel targeted therapies represents an opportunity to improve patient outcomes in TNBC.

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.

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.

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.

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.

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: 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]

Objective Currently, there is a disconnect between finding a patient’s relevant molecular profile and predicting actionable therapeutics. Here we develop and implement the Integrating Molecular Profiles with Actionable Therapeutics (IMPACT) analysis pipeline, linking variants detected from whole-exome sequencing (WES) to actionable therapeutics.Methods and materials The IMPACT pipeline contains 4 analytical modules: detecting somatic variants, calling copy number alterations, predicting drugs against deleterious variants, and analyzing tumor heterogeneity. We tested the IMPACT pipeline on whole-exome sequencing data in The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples with known EGFR mutations. We also used IMPACT to analyze melanoma patient tumor samples before treatment, after BRAF-inhibitor treatment, and after BRAF- and MEK-inhibitor treatment.Results IMPACT Food and Drug Administration (FDA) correctly identified known EGFR mutations in the TCGA lung adenocarcinoma samples. IMPACT linked these EGFR mutations to the appropriate FDA-approved EGFR inhibitors. For the melanoma patient samples, we identified NRAS p.Q61K as an acquired resistance mutation to BRAF-inhibitor treatment. We also identified CDKN2A deletion as a novel acquired resistance mutation to BRAFi/MEKi inhibition. The IMPACT analysis pipeline predicts these somatic variants to actionable therapeutics. We observed the clonal dynamic in the tumor samples after various treatments. We showed that IMPACT not only helped in successful prioritization of clinically relevant variants but also linked these variations to possible targeted therapies.Conclusion IMPACT provides a new bioinformatics strategy to delineate candidate somatic variants and actionable therapies. This approach can be applied to other patient tumor samples to discover effective drug targets for personalized medicine.IMPACT is publicly available at http://tanlab.ucdenver.edu/IMPACT.

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.

Cancer is a major public health problem in the United States and throughout the world. It is currently the second leading cause of death in the United States and is expected to surpass heart diseases in the next few years to become the leading cause of death [1]. The estimated number of new cases of invasive cancer (all types) in the United States is 1,658,370 which is equivalent of more than 4,500 new cancer diagnoses each day. In addition, the estimated number of deaths from cancer in 2015 is 589,430 corresponding to about 1,600 deaths per day [1]. Though there has been a steady increase in survival for most cancers the death rate remains unacceptable and for certain cancers i.e. lung and pancreatic cancers the 5-year relative survival is currently 18% and 7%, respectively. Traditional chemotherapy drugs act against all actively dividing cells (normal and cancerous cells) whereas targeted cancer therapies are drugs that interfere with specific molecular targets involved in cancer cell growth, progression and spread of cancer. Most targeted therapies are either small molecules or monoclonal antibodies. However, therapeutic strategies that target single molecular pathways eventually succumb to problems of intrinsic or acquired resistance due to extensive signaling “cross talk”. Thus, combination targeted therapies are more attractive, as they synergistically inhibit multiple receptors. However, overlapping toxicities and pharmacological interactions limit patient compliance, feasibility and efficacy. Clearly, there is an urgent need to develop new first-line agents with enhanced efficacy and reduced toxicity. We support the concept that the ideal drug maybe a broad spectrum drug whose efficacy is based not on the inhibition of a single target but rather a multi-targeted drug that affects several proteins or events that contribute to the etiology, pathogenesis and progression of diseases [2]. In addition, multipathway targeting is one of the strategies to overcome chemo-resistance. To design novel anticancer drugs with unique structural properties we have taken an innovative and nontraditional approach where we combine pharmacophoric components to create new and highly potent small molecules with a simple three component “A-B-C” structure where each pharmacophore is known to have anticancer properties on its own or when incorporated as a component of an existing drug. Our multi-component “A-B-C” drugs can target simultaneously two or more different molecular targets or molecular mechanisms in a single entity which should reduce the likelihood of drug resistance.

Introduction: Agents that reverse T cell inhibitory signals have reinvigorated the strategy of cancer immunotherapy and are leading to robust clinical responses. In colorectal cancer (CRC), objective responses to single-agent therapy with anti-PD-1/PD-L1 antibodies have largely been restricted to the small proportion of CRC patients with microsatellite unstable (MSI-high) disease. Although there are additional subsets of advanced CRC patients that are responsive to immune checkpoint inhibition in combination with tumor-targeted agents, preclinical models have been hampered by their immune-compromised status. In order to gain a better biological understanding of the context of immune responses and facilitate preclinical evaluation of combination strategies incorporating cancer immunotherapy, we developed a “hematopoietic” humanized mouse model utilizing patient-derived CRC xenografts with the intent of leveraging this model for the development of rational combinations. Methods: BRG.NODSirpalfa newborn pups were humanized through transplantation of 1x105 CD34+ cells purified from umbilical cord blood. Mice were evaluated for chimerism at 8 and 12 weeks. At 16 weeks, tumor tissue from established PDX models was implanted on the right and left flanks of humanized mice. When the average tumor size reached a volume of ~150-300 mm3, the mice were randomized into vehicle or nivolumab (30 mg/kg twice weekly i.p.) treatment groups according to %chimerism. Mice were monitored for signs of toxicity and tumor size was evaluated twice weekly by caliper measurements (tumor volume= (length × width2) × 0.52). At the end of the treatment, mice were euthanized while sera, lymph nodes, spleen, bone marrow and tumors were collected for immunological assessment. Results: As preliminary proof-of-concept, we successfully established 3 humanized CRC PDX models and one breast cancer cell line (MDA-MB-231). In one of the CRC (MSI-high) and in the breast model we observed tumor growth inhibition in the treated groups vs control. We detected differences in PD-1 expression among treated versus control mice, with lower expression in the nivolumab-treated groups. We also observed an increased number of TILs, CD8+ T cells and greater numbers of T cells in the lymph nodes of treated mice, suggesting T cell expansion. Mice were highly chimeric with high TILs whereas responder tumors exhibited an increase of CD44 high IFN+ T cells, high CD8% and a higher effector memory% (HLADR+, CD45RO+). Conclusions: Humanized PDX models were successfully established and tumor engraftment occurred in all humanized mice with nivolumab-treated mice demonstrating the development of lymph nodes that were populated by activated T cells. These preliminary results demonstrate that human immunity and PD-1 expressing T cells exist in these models and provide the basis for planned immunotherapy combination studies. Citation Format: Anna Capasso, Julie Lang, Todd M. Pitts, Sarah Lindsey Davis, Christopher Lieu, Scott Kopetz, Stacey Bagby, Paul Francoeur, John J. Tentler, Jill Slansky, Roberta Pelanda, Gail Eckhardt. Development and validation of humanized mice models implanted with patient derived colorectal cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1662. doi:10.1158/1538-7445.AM2017-1662

612 Background: Although CRC is rare in young adults, the incidence has increased recently and patients present more commonly with Stage III or IV disease which may reflect differing biology. Prior analyses revealed a statistically significant increase in the risk of death in young patients with metastatic CRC compared to older patients. Methods: To assess age-dependent genetic changes, DNA and RNA from younger ( < 40) and older ( > 65) CRC were isolated, and whole exome and transcriptome sequencing analysis from 4699 FFPE CRC clinical specimens were performed. Hybridization capture was performed on up to 3,769 exons from 403 cancer-related genes and 47 introns of 19 genes commonly rearranged in cancer. Samples were sequenced to high (average 688X) uniform coverage. CRC PDXs were injected into athymic nude mice and randomized into vehicle control, refametinib (MEKi), OMP-18R5 (monoclonal Ab against FZD 1, 2, 5, 7, and 8), or the combination and treated for at least 28 days. Results: Gene alteration rates in the younger and older cohorts were similar in a majority of the genes analyzed. Alterations in CTNNB1 [p < 0.001, False Discovery Rate (FDR) = 0.07] and MLH1 (p < 0.001, FDR = 0.017) were found to be more common in younger patients. Older patients were more likely to have alterations in APC (p < 0.01, FDR < 0.01) and FAM123B (p < 0.01, FDR = 0.03). The CTNNB1 data provided support to explore the WNT pathway as a potential target for younger patients. Therefore, we conducted an in vivo study of MEKi and OMP-18R5 to assess efficacy in molecularly defined young CRC PDX models. In a young PDX models, we observed enhanced combination effects with tumor growth inhibition index (treated/control) of 13% in the combination arm (compared to tumor growth of 150 %, and 116%, respectively with MEKi and OMP-18R5 alone). Interestingly, in this PDX models, RNA sequencing data showed baseline enrichment in the WNT and mTOR pathway, not observed in the older PDX models. Conclusions: The combination of refametinib and OMP-18R5 showed enhanced antitumor activity in combination compared to the single agent arms. Studies investigating predictive biomarkers of response are currently underway.

The goal of this study was to investigate the activity of the selective MEK1/2 inhibitor TAK-733 in both melanoma cell lines and patient-derived melanoma xenograft models. In vitro cell proliferation assays using the sulforhodamine B assay were conducted to determine TAK-733 potency and melanoma responsiveness. In vivo murine modeling with eleven patient-derived melanoma explants evaluated daily dosing of TAK-733 at 25 or 10 mg/kg. Immunoblotting was performed to evaluate on-target activity and downstream inhibition by TAK-733 in both in vitro and in vivo studies. TAK-733 demonstrated broad activity in most melanoma cell lines with relative resistance observed at IC50 > 0.1 μmol/L in vitro. TAK-733 also exhibited activity in 10 out of 11 patient-derived explants with tumor growth inhibition ranging from 0% to 100% (P < 0.001–0.03). Interestingly, BRAFV600E and NRAS mutational status did not correlate with responsiveness to TAK-733. Pharmacodynamically, pERK was suppressed in sensitive cell lines and tumor explants, confirming TAK-733–mediated inhibition of MEK1/2, although the demonstration of similar effects in the relatively resistant cell lines and tumor explants suggests that escape pathways are contributing to melanoma survival and proliferation. These data demonstrate that TAK-733 exhibits robust tumor growth inhibition and regression against human melanoma cell lines and patient-derived xenograft models, suggesting that further clinical development in melanoma is of scientific interest. Particularly interesting is the activity in BRAF wild-type models, where current approved therapy such as vemurafenib has been reported not to be active. Mol Cancer Ther; 14(2); 317–25. ©2014 AACR.

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

Purpose: Results from clinical trials involving resistance to molecularly targeted therapies have revealed the importance of rational single-agent and combination treatment strategies. In this study, we tested the efficacy of a type 1 insulin-like growth factor receptor (IGF1R)/insulin receptor (IR) tyrosine kinase inhibitor, OSI-906, in combination with a mitogen–activated protein (MAP)–ERK kinase (MEK) 1/2 inhibitor based on evidence that the MAP kinase pathway was upregulated in colorectal cancer cell lines that were resistant to OSI-906. Experimental Design: The antiproliferative effects of OSI-906 and the MEK 1/2 inhibitor U0126 were analyzed both as single agents and in combination in 13 colorectal cancer cell lines in vitro. Apoptosis, downstream effector proteins, and cell cycle were also assessed. In addition, the efficacy of OSI-906 combined with the MEK 1/2 inhibitor selumetinib (AZD6244, ARRY-142886) was evaluated in vivo using human colorectal cancer xenograft models. Results: The combination of OSI-906 and U0126 resulted in synergistic effects in 11 of 13 colorectal cancer cell lines tested. This synergy was variably associated with apoptosis or cell-cycle arrest in addition to molecular effects on prosurvival pathways. The synergy was also reflected in the in vivo xenograft studies following treatment with the combination of OSI-906 and selumetinib. Conclusions: Results from this study demonstrate synergistic antiproliferative effects in response to the combination of OSI-906 with an MEK 1/2 inhibitor in colorectal cancer cell line models both in vitro and in vivo, which supports the rational combination of OSI-906 with an MEK inhibitor in patients with colorectal cancer. Clin Cancer Res; 19(22); 6219–29. ©2013 AACR.

Purpose: The mitogen-activated protein kinase (MAPK) pathway is a crucial regulator of cell proliferation, survival, and resistance to apoptosis. MEK inhibitors are being explored as a treatment option for patients with KRAS-mutant colorectal cancer who are not candidates for EGFR-directed therapies. Initial clinical results of MEK inhibitors have yielded limited single-agent activity in colorectal cancer, indicating that rational combination strategies are needed. Experimental Design: In this study, we conducted unbiased gene set enrichment analysis and synthetic lethality screens with selumetinib, which identified the noncanonical Wnt/Ca++ signaling pathway as a potential mediator of resistance to the MEK1/2 inhibitor selumetinib. To test this, we used shRNA constructs against relevant WNT receptors and ligands resulting in increased responsiveness to selumetinib in colorectal cancer cell lines. Further, we evaluated the rational combination of selumetinib and WNT pathway modulators and showed synergistic antiproliferative effects in in vitro and in vivo models of colorectal cancer. Results: Importantly, this combination not only showed tumor growth inhibition but also tumor regression in the more clinically relevant patient-derived tumor explant (PDTX) models of colorectal cancer. In mechanistic studies, we observed a trend toward increased markers of apoptosis in response to the combination of MEK and WntCa++ inhibitors, which may explain the observed synergistic antitumor effects. Conclusions: These results strengthen the hypothesis that targeting both the MEK and Wnt pathways may be a clinically effective rational combination strategy for patients with metastatic colorectal cancer. Clin Cancer Res; 19(15); 4149–62. ©2013 AACR.

Introduction: Inhibiting the activity of the epidermal growth factor receptor (EGFR) with monoclonal antibodies has been utilized as a therapeutic strategy for patients with metastatic colorectal cancer (CRC), leading to improved clinical results alone and in combination with standard chemotherapy. Many systematic reviews and metanalyses were performed to better understand the role of EGFR inhibition in CRC, revealing that KRAS exon 2 mutations and furthermore exons 3 and 4 and NRAS exons 2, 3, and 4 were predictive of non-responsiveness to these agents. Concurrent with these results has been the development of immunotherapy targeting immune regulatory checkpoints such as CTLA-4 and PD-1 that have initiated a new era in the treatment of cancer. In order to gain a better biological understanding of the context of immune responses and facilitate preclinical evaluation of cancer immunotherapy, we developed a hematopoietic humanized mouse model utilizing patient-derived CRC xenograft tumor models to assess immune therapy for RAS mutant CRC. Not only could evaluation of humanized RAS mutant PDX models provide additional information on the potential for clinical activity of immune therapies, but could also improve the understanding of immune responses to RAS mutant cancers. We therefore hypothesize that humanized RAS mutant colorectal PDX models can be used to evaluate the preclinical activity of immune targeted agents for treatment of RAS mutant colorectal cancer. Methods: Humanized BRG mice developed from the BALB/cRag2-/-IL2Rγc-/- (BRG) strain which is known to accept human hematopoietic stem cells, have been used to enhance engraftment. BRG newborn pups were humanized through transplantation of approximately 1x105 CD34+ cells purified from umbilical cord blood. The mice were evaluated for chimerism at 8 and 12 weeks. At 14 weeks, tumor tissue from established PDX models were implanted on the right and left flank of humanized mice. The tumor was selected among a cluster within the “immune-enriched” subtype (C2) based upon the RNAseq characterization of the models. When the average tumor size reached a volume of approximately ~150-300 mm3, the mice were randomized into either vehicle or nivolumab treatment groups. Mice were monitored daily for signs of toxicity and weighed twice weekly. They were treated with nivolumab (30 mg/kg) twice a week by intraperitoneal injection for 15 days. Tumor size was evaluated twice weekly by caliper measurements using the following equation: tumor volume= (length × width2) × 0.52. At the end of the treatment, mice were euthanized while sera, lymph nodes, spleen, bone marrow and tumors were collected for further investigation. Results: Humanized RAS mutant CRC PDX models were successfully established in vivo. While no differences were observed in tumor growth among the control and treated arms, we were able to detect differences in PD1 expression among treated versus control mice, with lower expression in the nivolumab treated group. We also observed higher numbers of T cells in the lymph nodes of nivolumab treated mice, suggesting T cell expansion. Interestingly, we also observed an increase of T cells in the spleen and blood and late occupancy of T cells in the bone marrow. Two of the treated mice exhibited identifiable TILs that were comprised of a majority of CD4+ T cells with an activated phenotype (CD69+). Conclusions: Humanized KRAS mutant CRC PDX models were successfully established and tumor engraftment occurred in all humanized mice with nivolumab-treated mice demonstrating the development of lymph nodes that were populated by activated T cells. These preliminary results demonstrate that human immunity and PD-1 expressing T cells exist in these models and provide the basis for planned immunotherapy combination studies. This abstract is also being presented as Poster A23. Citation Format: Anna Capasso, Julie Lang, Todd M. Pitts, S. Lindsey Davis, Chris H. Lieu, Stacey M. Bagby, Aik Choon Tan, John J. Tentler, Jill E. Slansky, Roberta Pelanda, S. Gail Eckhardt. Characterizing the immune context of responses to immunotherapy in humanized patient derived xenograft models of CRC. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr PR03.

Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. Although recent therapeutic advances have prolonged patient survival, the prognosis remains dismal. C-MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that MERTK expression correlates with disease progression. MERTK expression was highest in metastatic melanomas, followed by primary melanomas, while the lowest expression was observed in nevi. Additionally, over half of melanoma cell lines overexpressed MERTK compared with normal human melanocytes; however, overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the MERTK ligand GAS6 resulted in the activation of several downstream signaling pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and diminished tumor volume by 60% in a human melanoma murine xenograft model. Treatment of melanoma cells with UNC1062, a novel MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of MERTK-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar, and inhibited invasion of melanoma cells. This work establishes MERTK as a therapeutic target in melanoma and provides a rationale for the continued development of MERTK-targeted therapies.

Purpose: The Aurora kinases are a family of conserved serine-threonine kinases with key roles in mitotic cell division. As with other promising anticancer targets, patient selection strategies to identify a responsive subtype will likely be required for successful clinical development of Aurora kinase inhibitors. The purpose of this study was to evaluate the antitumor activity of the Aurora and angiogenic kinase inhibitor ENMD-2076 against preclinical models of breast cancer with identification of candidate predictive biomarkers. Experimental Design: Twenty-nine breast cancer cell lines were exposed to ENMD-2076 and the effects on proliferation, apoptosis, and cell-cycle distribution were evaluated. In vitro activity was confirmed in MDA-MB-468 and MDA-MB-231 triple-negative breast cancer xenografts. Systematic gene expression analysis was used to identify up- and downregulated pathways in the sensitive and resistant cell lines, including within the triple-negative breast cancer subset. Results: ENMD-2076 showed antiproliferative activity against breast cancer cell lines, with more robust activity against cell lines lacking estrogen receptor expression and those without increased HER2 expression. Within the triple-negative breast cancer subset, cell lines with a p53 mutation and increased p53 expression were more sensitive to the cytotoxic and proapoptotic effects of ENMD-2076 exposure than cell lines with decreased p53 expression. Conclusions: ENMD-2076 exhibited robust anticancer activity against models of triple-negative breast cancer and the candidate predictive biomarkers identified in this study may be useful in selecting patients for Aurora kinase inhibitors in the future. Clin Cancer Res; 19(1); 291–303. ©2012 AACR.

Background: A plethora of agents is in early stages of development for colorectal cancer (CRC), including those that target the insulin-like growth factor I receptor (IGFIR) pathway. In the current environment of numerous cancer targets, it is imperative that patient selection strategies be developed with the intent of preliminary testing in the latter stages of phase I trials. The goal of this study was to develop and characterize predictive biomarkers for an IGFIR tyrosine kinase inhibitor, OSI-906, that could be applied in CRC-specific studies of this agent. Methods: Twenty-seven CRC cell lines were exposed to OSI-906 and classified according to IC50 value as sensitive (≤1.5 μmol/L) or resistant (>5 μmol/L). Cell lines were subjected to immunoblotting and immunohistochemistry for effector proteins, IGFIR copy number by fluorescence in situ hybridization, KRAS/BRAF/phosphoinositide 3-kinase mutation status, and baseline gene array analysis. The most sensitive and resistant cell lines were used for gene array and pathway analyses, along with shRNA knockdown of highly ranked genes. The resulting integrated genomic classifier was then tested against eight human CRC explants in vivo. Results: Baseline gene array data from cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which, in combination with IGFIR fluorescence in situ hybridization and KRAS mutational status, was able to predict with 100% accuracy a test set of patient-derived CRC xenografts. Conclusions: These results indicate that an integrated approach to the development of individualized therapy is feasible and should be applied early in the development of novel agents, ideally in conjunction with late-stage phase I trials. Clin Cancer Res; 16(12); 3193–204. ©2010 AACR.

Purpose: This in vivo study was designed to investigate the efficacy of ENMD-2076, a small-molecule kinase inhibitor with activity against the Aurora kinases A and B, and several other tyrosine kinases linked to cancer, including vascular endothelial growth factor receptor 2, cKit, and fibroblast growth factor receptor 1, against murine xenograft models of human colorectal cancer (CRC). Experimental Design: HT-29 CRC cell line xenografts were treated with either vehicle or ENMD-2076 (100 or 200 mg/kg) orally daily for 28 days. Tumor growth inhibition, dynamic contrast-enhanced magnetic resonance imaging, and 18FDG-positron emission tomography were conducted to assess the antiproliferative, antiangiogenic, and antimetabolic responses, respectively. Effects on proliferation were also analyzed by immunohistochemical methods. Additionally, three patient-derived xenografts from primary and metastatic sites were treated with ENMD-2076 (100 mg/kg) and assessed for tumor growth inhibition. Results: In the HT-29 xenograft model, ENMD-2076 induced initial tumor growth inhibition followed by regression. Treatment was associated with significant tumor blanching, indicating a loss of vascularity and substantial reductions in tumor vascular permeability and perfusion as measured by dynamic contrast-enhanced magnetic resonance imaging. Positron emission tomography scanning showed significant decreases in 18FDG uptake at days 3 and 21 of treatment, which was associated with a marked reduction in proliferation as assessed by Ki-67. All three of the patient-derived xenografts tested were sensitive to treatment with ENMD 2076 as measured by tumor growth inhibition. Conclusions: ENMD-2076 showed robust antitumor activity against cell line and patient-derived xenograft models of CRC that is detectable by functional imaging, supporting clinical investigation of this agent in CRC. Clin Cancer Res; 16(11); 2989–98. ©2010 AACR.

Background: Despite advances in targeted therapies for cancer, TNBC remains an aggressive breast cancer subtype with limited treatment options. Mutations in p53 are common in TNBC, however, the exact contribution of individual p53 mutations to response to therapy and mechanisms of acquired resistance are unknown. The purpose of this study was to characterize the activity of ENMD-2076, a multi-target Aurora kinase A and angiokinase inhibitor, against p53 mutated TNBC patient-derived tumor xenografts (PDX) and to identify differences in molecular pathways determining cellular fate in sensitive and resistant models. Methods: TNBC PDX models harboring different p53 mutations were used for ENMD-2076 treatment studies. Athymic nude mice were injected with tumor tissue and tumor volumes were measured twice a week. When the mean tumor volumes reached 150 mm3, mice were randomized and treated with vehicle control or ENMD-2076 200 mg/kg by oral gavage daily. A subset of animals were sacrificed at Day 4, 30, and at the time of acquired resistance for correlative tissue testing which included: immunofluorescence (IF) for p53, p73, BCL2, BAX, p16, phospho Aurora A (pAA) and phospho histone H3 (pHH3); immunohistochemistry (IHC) for cleaved caspase 3 (CC3) and Ki67; H&E and staining for senescence associated beta-galactosidase (SA -βgal) activity. Tumor growth inhibition (TGI) was calculated at Day 30 and sensitive models were treated until resistance when additional correlative tissue samples were obtained. Results: ENMD-2076 had significant anti-tumor activity against the CU_002 and CU_005 TNBC PDX models (TGI 71.3%, p value Conclusions: ENMD-2076 has pro-apoptotic anti-cancer activity in a subset of p53 mutated TNBC PDX models. Sensitivity was associated with the induction of p73, which may mediate the response in the absence of functional p53. Intrinsic and acquired resistance to ENMD-2076 in TNBC PTX models was associated with loss of p73 expression and an increase in markers associated with senescence, including p16 expression and SA β-gal activity. These data support the role of senescence as a potential mechanism of resistance to Aurora kinase inhibitors in p53 mutated TNBC and support the continued development of combination therapies including with inhibitors of pathways that mediate senescence. Citation Format: Anastasia A. Ionkina, S. Gail Eckhardt, Todd M. Pitts, Carol Sartorius, Peter Kabos, Jiyhe Kim, Aik Choon Tan, John J. Tentler, Jennifer R. Diamond. Senescence as a mechanism of resistance to the Aurora kinase and angiokinase inhibitor, ENMD-2076, in p53 mutated triple-negative breast cancer (TNBC) models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C34.

Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype defined by the lack of expression of ER, PR and HER2 over-expression. TNBC is heterogeneous in its 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 ENMD-2076, a selective Aurora kinase A inhibitor (AurKi). p53 and p73 knockdown (KD) models were developed to determine their role in mediating induction of apoptosis and senescence in response to AurK inhibition. Additionally, the effects of combining ENMD-2076 with the p53 modulator nutlin-3 were assessed. Methods: TNBC cell lines, MDA-MB-468 (p53 mut) and CAL51 (p53 WT) were transduced with shRNA constructs targeting p53 and p73 with > 80% knockdown as determined by RT-PCR. These clones were exposed to escalating doses of ENMD-2076 and the effect on proliferation was determined using an SRB assay. Proliferation was assessed using an SRB assay, and confirmed using a direct cell counting to control for increase in cell size following ENMD-2076 exposure. In the CAL51 KD lines, the effects of ENMD-2076 treatment on cellular senescence were determined using a senescence associated beta-galactosidase (SA β-gal) assay. Combination studies with nutlin-3 in MDA-MB-468 and HCC1937 were assessed by SRB and synergy measured using Calcusyn software. Results: KD of p53 and p73 in the MDA-MB-468p5310, MDA-MB-468p7355 cell lines resulted in an increase in the IC50 from 0.625 μM to 1.5 μM compared to scrambled. KD of CAL51 p53 and p73 increased from 0.16 µM to 0.41 µM, and 1.3 µM, in the CAL51scr, CAL51p5310, CAL51p7326, respectively. The sensitive MDA-MB-468 cell line (IC50 40 nM) and the sensitive CAL51 cell line (IC50 45 nM) were selected for further experimentation. Exposure to ENMD-2076 at concentrations of 0.5 μM and 1 μM for varying time points resulted in induction of senescence in the CAL51 cell line, but not in the MDA-MB-468 cell line, where induction of apoptosis at 48 hours was observed. In CAL51 p53 and p73 KD clones, induction of senescence was observed following exposure to ENMD-2076. Combination treatment with nutlin-3 showed synergistic activity. Conclusions: ENMD-2076 exhibited robust anticancer activity towards preclinical models of TNBC, in both p53 mutated and p53 WT cell lines, supporting future clinical investigation of AurKi in TNBC. In p53 WT TNBC, both p53 and p73 mediate sensitivity to ENMD-2076 and p73 is essential for induction of senescence following exposure to ENMD-2076. Synergistic anticancer activity of ENMD-2076 and nutlin reinforces the role of p53 in Aurora kinase inhibition of TNBC. Biomarkers predictive of response to ENMD-2076 in p53 mutated TNBC are being developed. These results with p53 KD cell lines are currently being confirmed in vivo. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A286. Citation Format: Anastasia A. Ionkina, John J. Tentler, Timothy P. Newton, Kelsey L. Brunkow, Jared S. Johnson, Aik Choon Tan, Todd M. Pitts, S. Gail Eckhardt, Jennifer R. Diamond. The role of p53 family tumor suppressors in mediating response to the Aurora and angiogenic kinase inhibitor ENMD-2076 in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A286.

Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. While recent therapeutic advances have prolonged patient survival, prognosis remains dismal. Mer is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that Mer expression correlates with disease progression. Mer expression was highest in metastatic melanomas followed by primary melanomas whereas the lowest expression was observed in nevi. In addition, over 50% of melanoma cell lines overexpressed Mer compared to normal human melanocytes, however overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the Mer ligand Gas6 resulted in activation of several downstream signaling pathways including MAPK/ERK, PI3K/Akt, and Jak/STAT. Mer inhibition via shRNA reduced Mer-mediated downstream signaling, reduced colony formation by up to 59% (p Citation Format: Jennifer Schlegel, Maria Sambade, Susan Sather, Stergios Moschos, Aik-Choon Tan, Amanda Winges, Deborah DeRyckere, Craig C. Carson, Dimitri G. Trembath, John J. Tentler, Gail Eckhardt, Pei-Fen Kuan, Ronald L. Hamilton, Lyn M. Duncan, C. Ryan Miller, Nana Nikolaishvili-Feinberg, Bentley R. Midkiff, Xiaodong Wang, Jing Liu, Weihe Zhang, Chao Yang, Stephen V. Frye, H. Shelton Earp, Janiel Shields, Douglas K. Graham. Mer receptor tyrosine kinase is a novel therapeutic target in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3037. doi:10.1158/1538-7445.AM2013-3037

Introduction: Triple-negative breast cancer (TNBC) is a clinically aggressive breast cancer subtype which lacks estrogen receptor expression. Histone deacetylase (HDAC) inhibitors have previously been shown to induce estrogen receptor expression in TNBC models. Paragazole is a selective class I HDAC inhibitor (HDACs 1, 2, and 3) currently in preclinical development. In this project, we explored the antiproliferative and proapoptotic activity of paragazole and investigated the combination of paragazole and chemotherapy in a panel of breast cancer cell lines. Methods: Breast cancer cell lines were exposed to varying sub-micromolar concentrations of paragazole alone and in combination with carboplatin, paclitaxel, and gemcitabine. Proliferation was assessed using an SRB assay and analyzed using the Calcusyn program, whereby synergy was defined as a Combination Index of less than 1. Cell cycle analysis was performed using flow cytometry and apoptosis was analyzed using a caspase 3/7 assay. Baseline HDAC expression was measured by RT-PCR and RNA-Seq was used pre- and post-treatment in a subset of 2 sensitive and 2 resistant cell lines. Results: Exposure to paragazole resulted in a decrease in cell proliferation at submicromolar concentrations with more robust antiproliferative activity observed in the TNBC cell lines compared to the estrogen receptor positive cell lines. An increase in apoptosis that was maximal at 24-48 hours was observed with single agent paragazole in a subset of sensitive TNBC cell lines. The combinations of paragazole with either paclitaxel, gemcitabine, or carboplatin all resulted in additive or synergistic growth inhibition. Expression of HDAC 1 was higher in the sensitive TNBC cell lines than in the resistant lines. Exposure to paragazole led to an increase in CARM1 mediated estrogen receptor expression. Discussion: These in vitro results demonstrate that paragazole has antiproliferative and proapototic activity against TNBC cell lines and may potentiate the activity of chemotherapy. These data support confirmation of the findings using in vivo models and the investigation of paragazole in combination with anti-estrogen agents such as tamoxifen and fulvestrant. Citation Format: David P. Astling, John J. Tentler, Benjamin R. Cross, Timothy P. Newton, Todd M. Pitts, Eric Gunther, Xuedong Liu, S. Gail Eckhardt, Jennifer R. Diamond. A novel class I histone deacetylase (HDAC) inhibitor, paragazole, demonstrates antiproliferative and proapoptotic effects in triple-negative breast cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1006. doi:10.1158/1538-7445.AM2013-1006

Alcohol abuse in men has been long known to result in testicular atrophy, impotence, and reduced reproductive function, and only recently have the mechanisms responsible for this clinical picture been elucidated. Although direct gonadal toxicity after ethanol (ETOH) exposure has been well documented, the failure of serum luteinizing hormone (LH) and serum follicle-stimulating hormone (FSH) levels to rise in the presence of blunted testosterone values and impaired spermatogenesis supports a central neuroendocrine defect as well. The neuroendocrine target of ethanol’s action appears to be mediated both at a pituitary and a supra-pituitary level. Alcohol also appears to be disruptive to the male growth hormone axis, resulting in suppression of both peripheral growth hormone (GH) and insulin-like growth factor I (IGF-I) levels, in addition to altering pituitary GH synthesis. This chapter, which will deal with male reproductive and growth dysfunction, will attempt to summarize the available data on this topic.