For xenograft experiments body weight (grams) are plotted over time (days) for different BVD-523 treatment groups in experiment using (A) A375 (n=15 for BVD-523 treatment groups, n= 10 for vehicle group), (B) Colo205 (n=12), (D) MIAPaCa2 (n=12), and (F) ZR-75.1 (n=15). Mean tumor volume {plus minus}SEM over time (days), is shown for (C) MIAPaCA2 and (E) ZR-75.1, following BVD-523 treatment.

In SW48 colorectal cells engineered with KRAS alleles, response to paclitaxel was unaltered.

Body Weight (grams) over time (days) is shown for treatment groups in xenograft derived from a vemurafenib-relapsed patient.

Supplementary materials and methods and supplementary figure legends

Using Reverse Phase Protein Array (RPPA), effects on proteins are measured in cell lines (A375, AN3Ca, Colo205, HCT116, HT29 and MIAPaCa2) following treatment with ERK1/2 inhibitors BVD-523 (BVD), Vx11e (Vx), GDC-0994 (GDC), or SCH772984 (SCH) (n=4 for each condition).

(A) FACS data for UACC-62 cell line following 200nM and 2000nM treatment with BVD-523 for 24 hours. (B) Percentage of MIAPaCa2 cells in G1, S, or G2 phase of the cell cycle following 0, 24 and 48 hours of 5μM BVD-523 treatment.

Combination interactions between BVD-523 and vemurafenib or dabrafenib were assessed in cell line (A) G361 and (B) A375 (n=3 for each). Growth inhibition and Loewe Excess is shown for 8 x 10 dose matrices. (C) BVD-523 in combination with dabrafenib markedly delays the onset of acquired resistance in A375 BRAFV600E melanoma cells.

(A) Percent change compared to DMSO treated control for following treatment with ERK inhibitors BVD-523 (BVD), Vx11 (Vx), GDC-0994 (GDC), and SCH772984 (SCH) are shown for pAkt (S473), pP70 S6K (S371 S394), pS6 Ribo Prot (S240 244) and pS6 Ribo Prot (S240 244), and (B) pBAD (S112), n=4 for each condition. (C) Western blot assays of cellular and nuclear fractions from RKO cell line following treatment with BVD-523, trametinib, SCH772984, or dabrafenib.

Herein, we report a novel series of highly potent and selective triazolothiadiazole c-Met inhibitors. Starting with molecule 5, we have applied structure-based drug design principles to identify the triazolothiadiazole ring system. We successfully replaced the metabolically unstable phenolic moiety with a quinoline group. Further optimization around the 5,6 bicyclic moiety led to the identification of 21. Compound 21 suffered from PDE3 selectivity issues and subsequent, structurally informed design led to the discovery of compound 23. Compound 23 has exquisite kinase selectivity, excellent potency, favorable ADME profile, and showed dose-dependent antitumor efficacy in a SNU-5 gastric cancer xenograft model.

The reported proof of principle study demonstrated the feasibility of local delivery of a c-Met inhibitor (VXc-140) in a subcutaneous xenograft tumor model. VXc-140 was formulated in a wafer delivery system for direct implantation into the tumor. Systemic and local tumor exposure of VXc-140 was analyzed. High tumor exposures coupled with fast release of compound were associated with significant tumor regression and reduction in tumor levels of phosphorylated c-Met. High VXc-140 tumor-to-plasma ratios (∼42 at the tumor periphery) were achieved. The tumor response achieved (7/11 partial response) with VXc-140 with the local delivery in the wafer (4 mg over 15 days) was comparable to the regression observed (11/15 partial response) for VXc-140 in the oral delivery (∼8 mg total administered once a day for 2 weeks). Notably, the plasma levels in animals implanted with VXc-140 wafers ranged from 2 to 4 μM, which, although higher than trough levels achieved with oral administration, were well below oral Cmax levels (∼42 μM) suggesting that toxicities associated with Cmax exposure may be reduced or eliminated by local delivery. The high tumor to plasma exposure of VXc-140 and the efficacy observed with local wafer delivery warrants further exploration into the utility of local delivery.

Abstract Background: The efficacy of chemotherapeutic agents such as doxorubicin, which cause lethal DNA double-strand breaks (DSBs), is diminished by efficient repair of the damaged DNA in cancer cells. DNA-PK is a key regulator of the non-homologous end joining (NHEJ) pathway, which is responsible for repairing DSBs. Studies of nonselective inhibitors of DNA-PK have shown that cancer cells depend on DNA-PK for survival following treatment with DSB-inducing agents. However, a comprehensive characterization of DNA-PK inhibition has been hampered by a lack of selective inhibitors. Here we describe VX-984, a potent and selective inhibitor of DNA-PK, and its preclinical profile in combination with doxorubicin both in vitro and in vivo. Methods: VX-984 was examined as a single agent and in combination with doxorubicin or pegylated liposomal doxorubicin (PLD) in a panel of breast cancer cell lines and in mouse xenograft models, respectively. Results: In vitro, inhibition of DNA-PK by VX-984 enhanced the cytotoxic activity of doxorubicin in established breast cancer cell lines and in primary ovarian tumor explants. Notably, mean Bliss DE >10% (strong synergy) were observed for doxorubicin in the presence of VX-984 in 22 of 35 breast cancer cell lines and 21 of 44 ovarian cancer cell lines in a broad cancer cell line screen. Further, the efficacy observed with VX-984 was associated with increased DNA damage as measured by phosphorylated histone H2AX (gamma-H2AX) and phosphorylated Kruppel-associated protein (pKAP1) in DU4475, MDA-MB-436 and MDA-MB-468 breast cancer cell lines, which is consistent with diminished DSB repair. In vivo, VX-984 significantly enhanced the efficacy of PLD in ovarian cancer patient-derived xenograft models and in cell line xenograft models. Conclusions: These data provide evidence that inhibition of DNA-PK by VX-984 enhances the efficacy of doxorubicin in preclinical models and support the use of VX-984 in combination with DSB agents such as anthracyclines including PLD for the treatment of breast and ovarian cancers. VX-984 is currently in a Phase 1 clinical trial in combination with PLD. Sponsored by Vertex Pharmaceuticals Incorporated. Citation Format: Boucher D, Newsome D, Takemoto D, Hillier S, Wang Y, Arimoto R, Maxwell J, Charifson P, Fields SZ, Tanner K, Penney MS. Preclinical characterization of VX-984, a selective DNA-dependent protein kinase (DNA-PK) inhibitor in combination with doxorubicin in breast and ovarian cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-06-05.

Background: The efficacy of chemotherapeutic agents such as doxorubicin, which cause lethal DNA double-strand breaks (DSBs), is diminished by efficient repair of the damaged DNA in cancer cells. DNA-PK is a key regulator of the non-homologous end joining (NHEJ) pathway, which is responsible for repairing DSBs. Studies of nonselective inhibitors of DNA-PK have shown that cancer cells depend on DNA-PK for survival following treatment with DSB-inducing agents. However, a comprehensive characterization of DNA-PK inhibition has been hampered by a lack of selective inhibitors. Here we describe VX-984, a potent and selective inhibitor of DNA-PK, and its preclinical profile in combination with doxorubicin both in vitro and in vivo. Methods: VX-984 was examined as a single agent and in combination with doxorubicin or pegylated liposomal doxorubicin (PLD) in a panel of breast cancer cell lines and in mouse xenograft models, respectively. Results: In vitro, inhibition of DNA-PK by VX-984 enhanced the cytotoxic activity of doxorubicin in established breast cancer cell lines and in primary ovarian tumor explants. Notably, mean Bliss DE u003e10% (strong synergy) were observed for doxorubicin in the presence of VX-984 in 22 of 35 breast cancer cell lines and 21 of 44 ovarian cancer cell lines in a broad cancer cell line screen. Further, the efficacy observed with VX-984 was associated with increased DNA damage as measured by phosphorylated histone H2AX (gamma-H2AX) and phosphorylated Kruppel-associated protein (pKAP1) in DU4475, MDA-MB-436 and MDA-MB-468 breast cancer cell lines, which is consistent with diminished DSB repair. In vivo, VX-984 significantly enhanced the efficacy of PLD in ovarian cancer patient-derived xenograft models and in cell line xenograft models. Conclusions: These data provide evidence that inhibition of DNA-PK by VX-984 enhances the efficacy of doxorubicin in preclinical models and support the use of VX-984 in combination with DSB agents such as anthracyclines including PLD for the treatment of breast and ovarian cancers. VX-984 is currently in a Phase 1 clinical trial in combination with PLD. Sponsored by Vertex Pharmaceuticals Incorporated. Citation Format: Boucher D, Newsome D, Takemoto D, Hillier S, Wang Y, Arimoto R, Maxwell J, Charifson P, Fields SZ, Tanner K, Penney MS. Preclinical characterization of VX-984, a selective DNA-dependent protein kinase (DNA-PK) inhibitor in combination with doxorubicin in breast and ovarian cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-06-05.

Abstract Aberrant activation of signaling through the RAS–RAF–MEK–ERK (MAPK) pathway is implicated in numerous cancers, making it an attractive therapeutic target. Although BRAF and MEK-targeted combination therapy has demonstrated significant benefit beyond single-agent options, the majority of patients develop resistance and disease progression after approximately 12 months. Reactivation of ERK signaling is a common driver of resistance in this setting. Here we report the discovery of BVD-523 (ulixertinib), a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and ERK1/2 selectivity. In vitro BVD-523 treatment resulted in reduced proliferation and enhanced caspase activity in sensitive cells. Interestingly, BVD-523 inhibited phosphorylation of target substrates despite increased phosphorylation of ERK1/2. In in vivo xenograft studies, BVD-523 showed dose-dependent growth inhibition and tumor regression. BVD-523 yielded synergistic antiproliferative effects in a BRAFV600E-mutant melanoma cell line xenograft model when used in combination with BRAF inhibition. Antitumor activity was also demonstrated in in vitro and in vivo models of acquired resistance to single-agent and combination BRAF/MEK–targeted therapy. On the basis of these promising results, these studies demonstrate BVD-523 holds promise as a treatment for ERK-dependent cancers, including those whose tumors have acquired resistance to other treatments targeting upstream nodes of the MAPK pathway. Assessment of BVD-523 in clinical trials is underway (NCT01781429, NCT02296242, and NCT02608229). Mol Cancer Ther; 16(11); 2351–63. ©2017 AACR.

This work demonstrates the application of a 3D culture system-Cells-in-Gels-in-Paper (CiGiP)-in evaluating the metabolic response of lung cancer cells to ionizing radiation. The 3D tissue-like construct-prepared by stacking multiple sheets of paper containing cell-embedded hydrogels-generates a gradient of oxygen and nutrients that decreases monotonically in the stack. Separating the layers of the stack after exposure enabled analysis of the cellular response to radiation as a function of oxygen and nutrient availability; this availability is dictated by the distance between the cells and the source of oxygenated medium. As the distance between the cells and source of oxygenated media increased, cells show increased levels of hypoxia-inducible factor 1-alpha, decreased proliferation, and reduced sensitivity to ionizing radiation. Each of these cellular responses are characteristic of cancer cells observed in solid tumors. With this setup we were able to differentiate three isogenic variants of A549 cells based on their metabolic radiosensitivity; these three variants have known differences in their metastatic behavior in vivo. This system can, therefore, capture some aspects of radiosensitivity of populations of cancer cells related to mass-transport phenomenon, carry out systematic studies of radiation response in vitro that decouple effects from migration and proliferation of cells, and regulate the exposure of oxygen to subpopulations of cells in a tissue-like construct either before or after irradiation.

Ionizing radiation (IR), which is widely used for the treatment of cancer, causes double-strand breaks (DSBs) in DNA. If left unrepaired, these DSBs are lethal to the cell. DNA-dependent protein kinase (DNA-PK) is a key enzyme in the non-homologous end joining (NHEJ) pathway that repairs DSBs caused by IR, or chemotherapeutic agents that cause DSBs such as doxorubicin. The goal of these studies was to characterize the radiation enhancing effects of VX-984, a selective and potent ATP-competitive inhibitor of the catalytic subunit of DNA-PK (DNA-PKcs), with a focus on non-small cell lung cancer (NSCLC) cells and tumor xenografts. VX-984 enhances the cytotoxicity of IR in a panel of cancer cell lines including NSCLC cell lines in vitro with dose enhancement factors (DEF) greater than 3. Notably, VX-984 combined with IR in normal human lung fibroblasts minimally enhanced the cytotoxicity compared to IR alone. Additionally, VX-984 decreased DNA-PKcs autophosphorylation on S2056 both in vitro and in vivo in NSCLC cells and attenuated the decay of the DNA damage markers γH2AX and pKAP1 in response to IR. In NSCLC PDX models VX-984, in combination with IR (2 Gy x 3), caused durable complete responses while IR alone only led to a delay in tumor growth, consistent with delayed DNA damage repair. In these models, the combination of VX-984 and IR was well tolerated. These data demonstrate that VX-984 is a potent radiation-enhancing agent and provide a strong rationale for the use of VX-984 in combination with IR for the treatment of NSCLC. Citation Format: Diane Boucher, Russell Hoover, Yuxin Wang, Yong Gu, David Newsome, Pamella Ford, Cameron Moody, Veronique Damagnez, Reiko Arimoto, Shawn Hillier, Mark Wood, William Markland, Brenda Eustace, Kevin Cottrell, Marina Penney, Brinley Furey, Kirk Tanner, John Maxwell, Paul Charifson. Potent radiation enhancement with VX-984, a selective DNA-PKcs inhibitor for the treatment of NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3716.

Abstract Non-small cell lung cancer (NSCLC) is a prevalent and deadly disease because of high incidence and relapse rates. One hypothesis for the high relapse rate is the existence of cancer stem cells (CSCs), a rare subpopulation of cells within these tumors, that are resistant to therapy and thought to be responsible for local and distal recurrence. CSCs are also able to self-renew and differentiate into multiple cell types forming the mass of new tumors. These differentiated progeny that constitute the bulk of the tumor are more sensitive to radiation and chemotherapeutic agents, express tissue of origin markers, and have an intrinsically limited lifespan. While most solid tumors have these properties, the degree of heterogeneity from patient to patient in the multi-lineage potential of the CSC population is not known. In particular, NSCLC CSC populations from primary patient tumors have not been well studied. The goal of this study was to characterize newly derived NSCLC CSC lines to determine the degree of heterogeneity between patient samples, and to provide a starting point for the discovery of novel differentiation therapeutic agents to target CSCs. We isolated six CSC lines from primary patient NSCLC tumors using an unbiased culture-based enrichment method, rather than a biased marker-based approach. Each of these six CSC lines was tumorigenic in immunodeficient mice at low cell numbers and the resulting tumors recapitulated the original tumor histology. All-trans retinoic acid (ATRA), a well-known differentiation agent for the treatment of acute promyelocytic leukemia (APL), maintains the normal growth and differentiation of human bronchial epithelial cells in culture. Each of the six CSC lines was treated with ATRA for two weeks to induce differentiation and then assessed for transcript and protein levels of candidate CSC and differentiation markers. Pre-treatment, the CSC lines all expressed CD44. Post-ATRA treatment, the majority of the CSC lines expressed Mucin-2, a marker of Goblet cell lineage. In addition, each cell line started to show evidence of the very early stages of differentiation into other lineages including Clara Cells, Neuroendocrine Cells, and Alveolar Type I and Type II cells. In one CSC line, ATRA treatment both in vitro and in vivo delayed tumor cell growth, induced the expression of Mucin-2 and decreased the expression of Nestin, a cancer stem cell marker. To further characterize CSC differentiation potential, we screened a pilot set of small molecules in two independent CSC lines to identify compounds that induce terminal differentiation, using Mucin-2 expression as readout. Surprisingly, despite the fact that these two CSC lines were from different histological subtypes (adenocarcinoma and squamous cell carcinoma) and displayed different transcriptional profiles post-differentiation, there was a good correlation between hit sets in the screens. Taken together, these data suggest that while the CSC of origin in tumors differs between patient samples in profile and function, there is promise to identify differentiation agents that are broadly active in different NSCLC subtypes. Further screening is currently being performed to identify these novel differentiation agents for therapeutic use. Citation Format: Dina Shlyakhter, Diane Boucher, Yong Gu, Amy B. Hall, Elaine Krueger, Anna Lindquist, Cheryl Murphy, Yuxin Wang, Mark Wood, Brenda Eustace. Differentiation screen identifies small molecules that target histologically divergent subtypes of patient-derived lung cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B24.