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Supplementary Data 3

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Supplementary Figure 1

Purpose:KRAS-mutant lung cancers have been recalcitrant to treatments including those targeting the MAPK pathway. Covalent inhibitors of KRAS p.G12C allele allow for direct and specific inhibition of mutant KRAS in cancer cells. However, as for other targeted therapies, the therapeutic potential of these inhibitors can be impaired by intrinsic resistance mechanisms. Therefore, combination strategies are likely needed to improve efficacy.Experimental Design: To identify strategies to maximally leverage direct KRAS inhibition we defined the response of a panel of NSCLC models bearing the KRAS G12C–activating mutation in vitro and in vivo. We used a second-generation KRAS G12C inhibitor, ARS1620 with improved bioavailability over the first generation. We analyzed KRAS downstream effectors signaling to identify mechanisms underlying differential response. To identify candidate combination strategies, we performed a high-throughput drug screening across 112 drugs in combination with ARS1620. We validated the top hits in vitro and in vivo including patient-derived xenograft models.Results:Response to direct KRAS G12C inhibition was heterogeneous across models. Adaptive resistance mechanisms involving reactivation of MAPK pathway and failure to induce PI3K–AKT pathway inactivation were identified as likely resistance events. We identified several model-specific effective combinations as well as a broad-sensitizing effect of PI3K-AKT–mTOR pathway inhibitors. The G12Ci+PI3Ki combination was effective in vitro and in vivo on models resistant to single-agent ARS1620 including patient-derived xenografts models.Conclusions:Our findings suggest that signaling adaptation can in some instances limit the efficacy of ARS1620 but combination with PI3K inhibitors can overcome this resistance.

Purpose:Effective targeted therapies are lacking for refractory and relapsed T-cell acute lymphoblastic leukemia (T-ALL). Suppression of the NOTCH pathway using gamma-secretase inhibitors (GSI) is toxic and clinically not effective. The goal of this study was to identify alternative therapeutic strategies for T-ALL.Experimental Design:We performed a comprehensive analysis of our high-throughput drug screen across hundreds of human cell lines including 15 T-ALL models. We validated and further studied the top hit, navitoclax (ABT-263). We used multiple human T-ALL cell lines as well as primary patient samples, and performed both in vitro experiments and in vivo studies on patient-derived xenograft models.Results:We found that T-ALL are hypersensitive to navitoclax, an inhibitor of BCL2 family of antiapoptotic proteins. Importantly, GSI-resistant T-ALL are also susceptible to navitoclax. Sensitivity to navitoclax is due to low levels of MCL-1 in T-ALL. We identify an unsuspected regulation of mTORC1 by the NOTCH pathway, resulting in increased MCL-1 upon GSI treatment. Finally, we show that pharmacologic inhibition of mTORC1 lowers MCL-1 levels and further sensitizes cells to navitoclax in vitro and leads to tumor regressions in vivo.Conclusions:Our results support the development of navitoclax, as single agent and in combination with mTOR inhibitors, as a new therapeutic strategy for T-ALL, including in the setting of GSI resistance.

Supplementary Table 1

Figures S1-S15 support the data in the main paper.

Legends to supplementary table, data and figures

SummaryTargeted therapeutics have advanced cancer treatment, but single agent activity remains limited by de novo and acquired resistance. Combining targeted drugs is broadly seen as a way to improve treatment outcome, motivating the ongoing search for efficacious combinations. To identify synergistic targeted therapy combinations and study the impact of tumor heterogeneity on combination outcome, we systematically tested over 5,000 two drug combinations at multiple doses across a collection of 81 non-small cancer cell lines. Both known and novel synergistic combinations were identified. Strikingly, very few combinations yield synergy across the majority of cell line models. Importantly, synergism mainly arises due to sensitization of single agent resistant models, rather than further sensitize already sensitive cell lines, frequently via dual targeting of a single or two highly interconnected pathways. This drug combinations resource, the largest of its kind should help delineate new synergistic regimens by facilitating the understanding of drug synergism in cancer.

MYCN-amplified neuroblastoma is a lethal subset of pediatric cancer. MYCN drives numerous effects in the cell, including metabolic changes that are critical for oncogenesis. The understanding that both compensatory pathways and intrinsic redundancy in cell systems exists implies that the use of combination therapies for effective and durable responses is necessary. Additionally, the most effective targeted therapies exploit an “Achilles’ heel” and are tailored to the genetics of the cancer under study. We performed an unbiased screen on select metabolic targeted therapy combinations and correlated sensitivity with over 20 subsets of cancer. We found that MYCN-amplified neuroblastoma is hypersensitive to the combination of an inhibitor of the lactate transporter MCT1, AZD3965, and complex I of the mitochondrion, phenformin. Our data demonstrate that MCT4 is highly correlated with resistance to the combination in the screen and lowly expressed in MYCN-amplified neuroblastoma. Low MCT4 combines with high expression of the MCT2 and MCT1 chaperone CD147 in MYCN-amplified neuroblastoma, altogether conferring sensitivity to the AZD3965 and phenformin combination. The result is simultaneous disruption of glycolysis and oxidative phosphorylation, resulting in dramatic disruption of adenosine triphosphate (ATP) production, endoplasmic reticulum stress, and cell death. In mouse models of MYCN-amplified neuroblastoma, the combination was tolerable at concentrations where it shrank tumors and did not increase white-blood-cell toxicity compared to single drugs. Therefore, we demonstrate that a metabolic combination screen can identify vulnerabilities in subsets of cancer and put forth a metabolic combination therapy tailored for MYCN-amplified neuroblastoma that demonstrates efficacy and tolerability in vivo.

MYCN-amplified neuroblastoma is a lethal subset of pediatric cancer. MYCN drives numerous effects in the cell, including metabolic changes that are critical for oncogenesis. The understanding that both compensatory pathways and intrinsic redundancy in cell systems exists implies that the use of combination therapies for effective and durable responses is necessary. Additionally, the most effective targeted therapies exploit an "Achilles' heel" and are tailored to the genetics of the cancer under study. We performed an unbiased screen on select metabolic targeted therapy combinations and correlated sensitivity with over 20 subsets of cancer. We found that MYCN-amplified neuroblastoma is hypersensitive to the combination of an inhibitor of the lactate transporter MCT1, AZD3965, and complex I of the mitochondrion, phenformin. Our data demonstrate that MCT4 is highly correlated with resistance to the combination in the screen and lowly expressed in MYCN-amplified neuroblastoma. Low MCT4 combines with high expression of the MCT2 and MCT1 chaperone CD147 in MYCN-amplified neuroblastoma, altogether conferring sensitivity to the AZD3965 and phenformin combination. The result is simultaneous disruption of glycolysis and oxidative phosphorylation, resulting in dramatic disruption of adenosine triphosphate (ATP) production, endoplasmic reticulum stress, and cell death. In mouse models of MYCN-amplified neuroblastoma, the combination was tolerable at concentrations where it shrank tumors and did not increase white-blood-cell toxicity compared to single drugs. Therefore, we demonstrate that a metabolic combination screen can identify vulnerabilities in subsets of cancer and put forth a metabolic combination therapy tailored for MYCN-amplified neuroblastoma that demonstrates efficacy and tolerability in vivo.

Purpose: KRAS-mutant lung cancers have been recalcitrant to treatments including those targeting the MAPK pathway. Covalent inhibitors of KRAS p.G12C allele allow for direct and specific inhibition of mutant KRAS in cancer cells. However, as for other targeted therapies, the therapeutic potential of these inhibitors can be impaired by intrinsic resistance mechanisms. Therefore, combination strategies are likely needed to improve efficacy. Experimental Design: To identify strategies to maximally leverage direct KRAS inhibition we defined the response of a panel of NSCLC models bearing the KRAS G12C–activating mutation in vitro and in vivo. We used a second-generation KRAS G12C inhibitor, ARS1620 with improved bioavailability over the first generation. We analyzed KRAS downstream effectors signaling to identify mechanisms underlying differential response. To identify candidate combination strategies, we performed a high-throughput drug screening across 112 drugs in combination with ARS1620. We validated the top hits in vitro and in vivo including patient-derived xenograft models. Results: Response to direct KRAS G12C inhibition was heterogeneous across models. Adaptive resistance mechanisms involving reactivation of MAPK pathway and failure to induce PI3K–AKT pathway inactivation were identified as likely resistance events. We identified several model-specific effective combinations as well as a broad-sensitizing effect of PI3K-AKT–mTOR pathway inhibitors. The G12Ci+PI3Ki combination was effective in vitro and in vivo on models resistant to single-agent ARS1620 including patient-derived xenografts models. Conclusions: Our findings suggest that signaling adaptation can in some instances limit the efficacy of ARS1620 but combination with PI3K inhibitors can overcome this resistance.

Purpose: Effective targeted therapies are lacking for refractory and relapsed T-cell acute lymphoblastic leukemia (T-ALL). Suppression of the NOTCH pathway using gamma-secretase inhibitors (GSI) is toxic and clinically not effective. The goal of this study was to identify alternative therapeutic strategies for T-ALL. Experimental Design: We performed a comprehensive analysis of our high-throughput drug screen across hundreds of human cell lines including 15 T-ALL models. We validated and further studied the top hit, navitoclax (ABT-263). We used multiple human T-ALL cell lines as well as primary patient samples, and performed both in vitro experiments and in vivo studies on patient-derived xenograft models. Results: We found that T-ALL are hypersensitive to navitoclax, an inhibitor of BCL2 family of antiapoptotic proteins. Importantly, GSI-resistant T-ALL are also susceptible to navitoclax. Sensitivity to navitoclax is due to low levels of MCL-1 in T-ALL. We identify an unsuspected regulation of mTORC1 by the NOTCH pathway, resulting in increased MCL-1 upon GSI treatment. Finally, we show that pharmacologic inhibition of mTORC1 lowers MCL-1 levels and further sensitizes cells to navitoclax in vitro and leads to tumor regressions in vivo. Conclusions: Our results support the development of navitoclax, as single agent and in combination with mTOR inhibitors, as a new therapeutic strategy for T-ALL, including in the setting of GSI resistance.

MYCN-amplified neuroblastoma is responsible for ~10% of all cancer-related deaths in the pediatric population. MYCN drives numerous effects in the cell, including metabolic changes that are critical for oncogenesis. The understanding that both compensatory pathways and intrinsic redundancy in cell systems exists implies that the use of combination therapies for effective and durable responses is necessary. Additionally, the most effective targeted therapies exploit an “Achilles’ heels” and are tailored to the genetics of the cancer under study. We performed an unbiased screen on select metabolic targeted therapy combinations and correlated sensitivity with over 20 subsets of cancer. We found that MYCN-amplified neuroblastoma are hypersensitive to the combination of AZD3965, an inhibitor of the lactate transporter MCT1, and phenformin, an inhibitor of complex I of the mitochondrion. Our data demonstrate MCT4 is highly correlated with resistance to the combination of AZD3965 and phenformin through the screen, is heavily methylated, and lowly expressed in MYCN-amplified neuroblastoma. Low MCT4 combines with high expression of the MCT2 and MCT1 chaperone CD147 in MYCN-amplified neuroblastoma, altogether conferring sensitivity to AZD3965 and phenformin combination. Metabolomic analysis showed that the result is simultaneous disruption of glycolysis and oxidative phosphorylation, resulting in dramatic disruption of adenosine triphosphate (ATP) production, endoplasmic reticulum (ER) stress, and apoptosis, which was seen through cell viability assays and protein analysis. In mouse models of MYCN-amplified neuroblastoma, the combination was tolerable at concentrations where it shrank tumors and did not increase white blood cell toxicity compared to single-drugs. Therefore, we demonstrate a metabolic combination screen can identify vulnerabilities in subsets of cancer and put forth a metabolic combination therapy tailored for MYCN-amplified neuroblastoma that demonstrates efficacy and tolerability in vivo. Citation Format: Krista M. Powell, Timonthy L. Lochmann, Konstantinos V. Floros, Marissa L. Calbert, Richard Kurupi, Giovanna T. Stein, Joseph McClanaghan, Ellen Murchie, Regina K. Egan, Patricia Greninger, Mikhail Dozmorov, Sivapriya Ramamoorthy, Madhavi Puchalapalli, Bin Hu, Lisa Shock, Jennifer Koblisnski, John Glod, Sosipatros A. Boikos, Cyril H. Benes, Anthony C. Faber. Catastrophic ATP loss underlines a metabolic combination therapy tailored for MYCN-amplified neuroblastoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-024.

The prosurvival BCL2 family member MCL1 is frequently dysregulated in cancer. To overcome the significant challenges associated with inhibition of MCL1 protein–protein interactions, we rigorously applied small-molecule conformational restriction, which culminated in the discovery of AMG 176, the first selective MCL1 inhibitor to be studied in humans. We demonstrate that MCL1 inhibition induces a rapid and committed step toward apoptosis in subsets of hematologic cancer cell lines, tumor xenograft models, and primary patient samples. With the use of a human MCL1 knock-in mouse, we demonstrate that MCL1 inhibition at active doses of AMG 176 is tolerated and correlates with clear pharmacodynamic effects, demonstrated by reductions in B cells, monocytes, and neutrophils. Furthermore, the combination of AMG 176 and venetoclax is synergistic in acute myeloid leukemia (AML) tumor models and in primary patient samples at tolerated doses. These results highlight the therapeutic promise of AMG 176 and the potential for combinations with other BH3 mimetics. Significance: AMG 176 is a potent, selective, and orally bioavailable MCL1 inhibitor that induces a rapid commitment to apoptosis in models of hematologic malignancies. The synergistic combination of AMG 176 and venetoclax demonstrates robust activity in models of AML at tolerated doses, highlighting the promise of BH3-mimetic combinations in hematologic cancers. See related commentary by Leber et al., p. 1511. This article is highlighted in the In This Issue feature, p. 1494

Aim: We investigated whether p53 activation with ALRN-6924 can prevent or reduce chemotherapy-induced hematopoietic toxicity while preserving or enhancing anti-tumor efficacy of chemotherapy in p53-mutant tumors. Materials and methods: ALRN-6924 is a clinical-stage, first-in-class, stabilized cell-permeating alpha-helical peptide that disrupts the interaction of the p53 tumor suppressor protein with its endogenous inhibitors, MDMX and MDM2. For p53 wild-type cells such as normal bone marrow, p53 activation can induce transient, dose-dependent cell cycle arrest, reducing sensitivity to chemotherapy-induced cellular toxicity. For p53-mutant cancer cells, ALRN-6924 has no effect on the cell cycle, leaving them vulnerable to chemotherapy. ALRN-6924-induced cell cycle arrest was measured by flow cytometry in human bone marrow CD34+ cells following incubation with ALRN-6924 ex vivo for 24 hours. DNA synthesis and DNA content were quantified by flow cytometry using EdU incorporation and Hoechst 33342 staining, respectively. Cell cycle arrest in the bone marrow of ALRN-6924-treated C57BL/6 mice was measured by flow cytometry using EdU incorporation in lineage negative, Kit positive hematopoietic stem and progenitor cells. Topotecan-induced DNA damage was measured in human bone marrow CD34+ cells by H2γX incorporation following exposure to vehicle or ALRN-6924 for 24 hours to induce cell cycle arrest, then incubated with topotecan for an additional 24 hours following a wash-out step. Topotecan-induced neutropenia was measured in female C57BL/6 mice following topotecan treatment on days 1-5 and either ALRN-6924 or vehicle on days 0-4. Female C57BL/6 mice bearing subcutaneous p53-mutant MC38 syngeneic tumors were treated with ALRN-6924, vehicle and topotecan on the same dosing regimen and followed until tumors reached >1000mm3. Results: ALRN-6924 induces transient, reversible cell cycle arrest in bone marrow cells in vitro and in vivo, and protects human bone marrow cells against topotecan-induced DNA damage ex vivo. In a mouse model of topotecan-induced neutropenia, ALRN-6924 protected against neutrophil depletion when daily administration started 24 hours prior to the 1st dose and 30 minutes before each subsequent dose of topotecan. ALRN-6924 does not diminish topotecan’s anti-tumor activity in the p53-mutant MC38 syngeneic mouse cancer model, with the ALRN-6924 + topotecan combination yielding modest enhancement of survival. Body weights and mortality data suggest ALRN-6924 and combinations with topotecan were tolerated at the doses tested. Conclusions: ALRN-6924 reduces chemotherapy-induced hematopoietic toxicity in healthy human bone marrow cells ex vivo and in mouse models of topotecan-induced neutropenia in vivo, while preserving or enhancing anti-tumor efficacy in p53-mutant tumors when administered intravenously prior to chemotherapy. These results support the first ALRN-6924 clinical trial for myelopreservation in topotecan-treated small-cell lung cancer patients (NCT04022876). Additional studies are underway to support ALRN-6924 as a tumor type-agnostic myelopreservation agent for cancer patients with tumors bearing p53 mutations who are treated with chemotherapy. Citation Format: Luis A Carvajal, David Sutton, Mariam Mounir, Joseph McClanaghan, Vincent Guerlavais, Manuel Aivado, Vojislav Vukovic, Allen Annis. The investigational peptide drug ALRN-6924, a dual inhibitor of MDMX and MDM2, is an effective myelopreservation agent [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C064. doi:10.1158/1535-7163.TARG-19-C064