Exploiting MCL-1 Dependency with Combination MEK + MCL-1 Inhibitors Leads to Induction of Apoptosis and Tumor Regression in KRAS Mutant Non-Small Cell Lung Cancer

Author Manuscript Published OnlineFirst on September 25, 2018; DOI: 10.1158/2159-8290.CD-18-0277 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Title: Exploiting MCL-1 dependency with combination MEK + MCL-1 inhibitors leads to induction of apoptosis and tumor regression in KRAS mutant non-small cell lung cancer

Varuna Nangia1*, Faria M. Siddiqui1*, Sean Caenepeel2, Daria Timonina1, Samantha J. Bilton1, Nicole Phan1, Maria Gomez-Caraballo1, Hannah L. Archibald1, Chendi Li1, Cameron Fraser3, Diamanda Rigas2, Kristof Vajda1, Lorin A. Ferris1, Michael Lanuti4, Cameron D. Wright4, Kevin A. Raskin5, Daniel P. Cahill6, John H. Shin6, Colleen Keyes7, Lecia V. Sequist8,9, Zofia Piotrowska8,9, Anna F. Farago8,9, Christopher G. Azzoli8,9, Justin F. Gainor8,9, Kristopher A. 3 10 2 1,9 2 Sarosiek , Sean P. Brown , Angela Coxon , Cyril H. Benes , Paul E. Hughes , Aaron N. Hata1,8,9

Affiliations: 1 Massachusetts General Hospital Cancer Center, Charlestown, MA 2 Department of Oncology Research, Amgen, Thousand Oaks, CA 3Department of Environmental Health, Harvard T. H. Chan School of Public Health, Boston, MA 4Department of Surgery, Massachusetts General Hospital, Boston, MA 5Department of Orthopaedics, Massachusetts General Hospital, Boston, MA 6Department of Neurosurgery, Massachusetts General Hospital, Boston, MA 7Division of Pulmonary and Critical Care Medicine, Department of Medicine, Massachusetts General Hospital, Boston, MA 8Division of Hematology Oncology, Department of Medicine, Massachusetts General Hospital, Boston, MA 9Department of Medicine, Harvard Medical School, Boston, MA 10Department of Medicinal Chemistry, Amgen, Thousand Oaks, CA. *equal contribution

Address correspondence to: Aaron N. Hata, Massachusetts General Hospital Cancer Center, 149 13th Street, Charlestown, MA 02129, USA. E-mail: [email protected].

Running Title: Combination MEK + MCL-1 inhibitors for KRAS mutant NSCLC

Conflict of Interest: A.N.H. receives research support from Amgen, Novartis and Relay Therapeutics. S.C., D.R., S.B., A.C. and P.E.H. are employees of Amgen, as noted in the affiliations.

Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 25, 2018; DOI: 10.1158/2159-8290.CD-18-0277 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract BH3 mimetic drugs, which inhibit pro-survival BCL-2 family proteins, have limited single-agent activity in solid tumor models. The potential of BH3 mimetics for these cancers may depend on their ability to potentiate the apoptotic response to chemotherapy and targeted therapies. Using a novel class of potent and selective MCL-1 inhibitors, we demonstrate that concurrent MEK + MCL-1 inhibition induces apoptosis and tumor regression in KRAS mutant non-small cell lung cancer (NSCLC) models, which respond poorly to MEK inhibition alone. Susceptibility to BH3 mimetics that target either MCL-1 or BCL-XL was determined by the differential binding of pro- apoptotic BCL-2 proteins to MCL-1 or BCL-XL, respectively. The efficacy of dual MEK + MCL-1 blockade was augmented by prior transient exposure to BCL-XL inhibitors, which promotes the binding of pro-apoptotic BCL-2 proteins to MCL-1. This suggests a novel strategy for integrating BH3 mimetics that target different BCL-2 family proteins for KRAS mutant NSCLC.

Statement of Significance Defining the molecular basis for MCL-1 versus BCL-XL dependency will be essential for effective prioritization of BH3 mimetic combination therapies in the clinic. We discover a novel strategy for integrating BCL-XL and MCL-1 inhibitors to drive and subsequently exploit apoptotic dependencies of KRAS mutant NSCLCs treated with MEK inhibitors.

1 Introduction 2 3 KRAS, a small GTPase that activates MAPK signaling, is one of the most frequently mutated 4 driver oncogenes (1). KRAS mutations, which are largely localized to codons G12, G13 and 5 Q61, decrease either intrinsic and/or GAP-mediated hydrolysis, causing constitutive activation of 6 MAPK and other downstream signaling pathways (2). Although effective molecular targeted 7 therapies that inhibit oncogenic mutant kinases in the RAS-MAPK pathway have been developed 8 (e.g. EGFR inhibitors for EGFR mutant NSCLC, BRAF inhibitors for BRAF mutant melanoma), 9 there currently are no approved targeted therapies for KRAS mutant cancers. KRAS mutations are 10 found in 20-25% of patients with non-small cell lung cancer (NSCLC) and predict for lack of 11 response to EGFR inhibitors (3). Attempts to target downstream MAPK signaling with inhibitors 12 of MEK1/2 have yielded disappointing results (4, 5), and strategies that simultaneously target 13 multiple signaling pathways have been limited by toxicity (6, 7). Most recently, a novel class of 14 KRAS inhibitors that covalently bind to the G12C mutant have been described (8, 9), although 15 these have yet to be tested in the clinic. Thus there remains an urgent need for new therapeutic 16 strategies that can target KRAS mutant cancers. 17 18 Several studies have shown that suppression of MAPK signaling, either by depletion of mutant 19 KRAS or by pharmacologic inhibition of downstream MEK1/2, is insufficient to induce 20 apoptosis in a significant number of KRAS mutant cell lines (10-12). Therapeutic strategies that 21 co-target kinase signaling pathways and apoptotic regulators may increase apoptosis and convert 22 cytostatic responses into tumor regressions (13). Activated kinase signaling pathways such as 23 MAPK (RAS/RAF/MEK/ERK) and PI3K/AKT converge on the BCL-2 protein family, which 24 regulates the mitochondrial or intrinsic apoptotic response (14). In cells with MAPK activation, 25 ERK phosphorylation suppresses the pro-apoptotic BH3 protein BIM by targeting it for 26 degradation (15, 16). MEK inhibition causes BIM to accumulate (16), however BIM can be 27 neutralized by pro-survival BCL-2 family members such as BCL-XL or MCL-1. Combining 28 MEK inhibitors with the BH3 mimetic navitoclax (ABT-263), which prevents the binding of 29 BIM to BCL-2 and BCL-XL, led to greater apoptosis and tumor regression in KRAS 30 experimental models compared to MEK inhibitors alone (11), and a clinical trial evaluating this 31 combination is currently on-going (NCT02079740, www.clinicaltrials.gov). To date, these

32 approaches have been limited to targeting BCL-2 and BCL-XL due to the lack of selective and 33 potent inhibitors that target other members of the BCL-2 family. 34 35 MCL-1 is frequently amplified in lung cancers (17), and the development of potent and selective 36 MCL-1 inhibitors has long been of interest. Recently, a novel MCL-1 inhibitor S63845 with in 37 vivo activity was reported (18). Significant activity was observed in leukemia, myeloma and 38 lymphoma models, and several different MCL-1 inhibitors are now currently in clinical 39 development for these malignancies (NCT02992483, NCT02979366, NCT02675452, 40 www.clinicaltrials.gov). Single agent activity of S63845 was limited in solid tumor models 41 including NSCLC and breast cancers, however combining S63845 with relevant kinase inhibitors 42 led to decreased cell viability of BRAF, EGFR and HER2-addicted cell lines in vitro, providing 43 proof of principle that MCL-1 inhibition, similar to BCL-XL inhibition, may potentiate the 44 response to kinase inhibitor targeted therapies. However, due to the lack of studies that directly 45 compare analogous combination strategies that target either MCL-1 or BCL-XL, the optimal 46 pairing of kinase inhibitors with BH3 mimetics that target different BCL-2 family proteins in 47 specific subsets of cancer remains undefined. 48 49 Here, we assessed the activity of a novel class of potent and selective spiro-macrocyclic MCL-1 50 inhibitors in combination with MEK inhibition in KRAS mutant NSCLC models and compared 51 this to the parallel strategy of MEK + BCL-XL inhibition. Distinct but overlapping subsets of 52 KRAS mutant NSCLC models were more sensitive to MEK + MCL-1 versus MEK + BCL-XL 53 inhibition, which was determined by the binding interactions between specific BCL-2 family 54 proteins. By altering the cellular localization and interactions between BCL-2 family proteins 55 with transient exposure to BCL-XL inhibitors, KRAS mutant NSCLC cells could be induced into 56 an MCL-1 dependent state with increased sensitivity to MEK + MCL-1 inhibition. These results 57 provide rationale for the clinical evaluation of MEK + MCL-1 inhibitors in KRAS mutant 58 NSCLC and suggest a broader strategy for integrating MCL-1 and BCL-XL inhibitors to 59 maximize efficacy of kinase inhibitor targeted therapies. 60 61

62 Results 63 64 Combination drug screen identifies synergistic activity between AM-8621 and MAPK pathway 65 inhibitors in cell lines with MAPK pathway activation. 66 67 Recently developed potent and selective MCL-1 inhibitors have demonstrated limited single- 68 agent activity against solid tumor malignancies (18, 19) suggesting that drug combinations may 69 be required to maximize the potential clinical benefit of this class of inhibitors for these cancers. 70 In order to determine agents that synergistically enhance the activity of MCL-1 inhibition, 187 71 compounds targeting a diverse array of mechanisms (Supplemental Table 1) were screened in 72 pair-wise combinations with a novel potent and selective MCL-1 inhibitor AM-8621 against a 73 panel of 15 diverse solid tumor cancer cell lines that had a range of sensitivities to AM-8621 74 (Supplemental Figure 1A-D). AM-8621 is the prototypical member of a novel class of spiro- 75 macrocyclic MCL-1 inhibitors, which also includes the tool compound AM-4907 and the clinical 76 compound AMG 176, that exhibit potent and selective MCL-1 inhibition in vitro and in vivo 77 (19). Synergistic drug combinations were determined using a Loewe excess additivity model 78 (20). A number of potential clinically relevant synergistic combinations were identified including 79 conventional cytotoxic chemotherapeutic agents, HSP90 inhibitors and BH3 mimetics targeting 80 BCL-XL/BCL-2 (Figure 1A, Supplemental Figure 1E). 81 82 To prioritize clinically promising MCL-1 inhibitor combinations, we considered the spectrum of 83 oncogenic driver mutations present in the cell lines. One of the most notable genotype associated 84 synergies was between AM-8621 and drugs that inhibit the MAPK pathway (MEK and BRAF 85 inhibitors) in cell lines with oncogenic MAPK pathway activation (Figure 1B). Six out of fifteen 86 cell lines showed statistically significant synergy with a majority of the MAPK pathway 87 inhibitors (MEK, BRAF) included in the screen, and eight cell lines showed statistically 88 significant synergy with a majority of MEK inhibitors. Of these eight cell lines, five harbored 89 mutations known to cause MAPK pathway activation (A427, H23 - KRAS; H1395, G-361 - 90 BRAF; H1437 - MEK1). Additionally, two of the three other cell lines that did not harbor 91 MAPK-activating mutations have previously been demonstrated to have functional MAPK 92 kinase pathway activation (HCC70 - triple negative breast cancer with activated RAS gene

93 signature (21); G402 - malignant rhabdoid tumor with hyperactivation of FGFR1 (22)). We next 94 confirmed synergy between AM-8621 and PD032591 (MEK inhibitor) and vemurafenib (BRAF 95 inhibitor), as well as the clinically relevant MEK inhibitor trametinib and BRAF inhibitor 96 dabrafenib, in KRAS and BRAF mutant cells by testing the heterologous (AxB) combinations or 97 corresponding self-crosses (AxA, BxB) for all possible dose combinations in a 10x10 complete 98 combination matrix. Strong synergy was again observed between AM-8621 and the MEK 99 inhibitors, PD0325901 and trametinib, in A427 and H23 cells (KRAS mutant NSCLC) and G-361 100 cells (BRAF mutant melanoma); synergy was not observed in the KRAS wild-type H661 101 (NSCLC) or DMS-273 (SCLC) cell lines (Figure 1C). Synergy was also observed between AM- 102 8621 and the BRAF inhibitors dabrafenib and vemurafenib in G-361 cells (Figure 1C). No 103 synergy was observed with the corresponding self-crosses. These results suggest that combining 104 MEK + MCL-1 inhibitors may be a promising combination strategy specifically for cancers with 105 MAPK pathway activation. 106 107 KRAS mutant NSCLC are sensitive to combination MEK + MCL-1 or BCL-XL inhibitors 108 109 Recent studies have demonstrated that combining MEK inhibitors with BH3 mimetics targeting 110 BCL-XL/BCL-2 (e.g. navitoclax or ABT-263) synergistically induces apoptosis in KRAS mutant 111 cancer models (11, 12). Based on the results of the combination drug screen, we hypothesized 112 that a parallel strategy targeting both MEK and MCL-1 might be effective against KRAS mutant 113 cancers, particularly those that do not respond to MEK + BCL-XL inhibition. To assess the 114 relative dependency of KRAS mutant cell lines on MCL-1 and BCL-XL, we first tested whether 115 AM-8621, navitoclax, or venetoclax (ABT-199, a selective BCL-2 inhibitor) alone could 116 decrease cell viability. Whereas most KRAS mutant colorectal cancer (CRC) cell lines were more 117 sensitive to navitoclax, subsets of KRAS mutant NSCLC cell lines were partially sensitive to 118 either AM-8621 or navitoclax, both or neither (Supplemental Figure 2A-C). Very little activity 119 of venetoclax was observed in either NSCLC or CRC cell lines. 120 121 We next sought to determine the degree of heterogeneity in the response of KRAS mutant 122 NSCLC cells to combined MEK + MCL-1 inhibition. Strong synergy was observed with the 123 combination of AM-8621 and trametinib in KRAS mutant NSCLC cell lines with partial

124 sensitivity to AM-8621 alone (e.g. H23, H2030), and the combination of navitoclax and 125 trametinib was synergistic in cell lines with partial sensitivity to navitoclax alone (e.g. H1734, 126 H1944; Figure 2A, Supplemental Figure 2D). Across the entire KRAS mutant NSCLC and CRC 127 cell line panels, navitoclax strongly enhanced the inhibitory effect of trametinib in almost all 128 colorectal cell lines (Supplemental Figure 2E), whereas the response of KRAS mutant NSCLC 129 cell lines was heterogeneous, with subsets of cell lines exhibiting enhancement with AM-8621, 130 navitoclax, both or neither (Figure 2B). Notably, significant activity of the AM-8621 + 131 trametinib combination was observed in cell lines that were completely insensitive to AM-8621 132 alone (e.g. A549, LU-99A). This heterogeneity was reflected in the degree of caspase-3/7 133 activation and apoptosis induced by MEKi + BH3 mimetic combinations (Supplemental Figure 134 2F, Figure 2C) and overall decrease in cell viability (Figure 2D). The apoptotic effect of AM- 135 8621 in combination with trametinib was recapitulated by siRNA-mediated MCL-1 knockdown 136 (Supplemental Figure 2G), validating an on-target mechanism of MCL-1 inhibition by AM- 137 8621. We also confirmed that the effects of navitoclax were due primarily to inhibition of BCL- 138 XL, as similar results were obtained using the BCL-XL selective inhibitor A1331852 (23) but 139 not the BCL-2 selective inhibitor venetoclax (Supplemental Figure 2H). These data demonstrate 140 that inhibition of MCL-1 or BCL-XL potentiates the effects of MEK inhibition in distinct but 141 overlapping subsets of KRAS mutant NSCLC. 142 143 Sensitivity to MCL-1 and BCL-XL inhibition is determined by specific BCL-2 family protein 144 interactions 145 146 Given the potential heterogeneity of response of KRAS mutant NSCLCs to MEK inhibitor + BH3 147 mimetic combinations, a more complete understanding of the underlying apoptotic dependencies 148 that determine drug sensitivity will be critical for selecting the most effective combination. 149 Profiling the response to AM-8621 across a large cancer cell line panel demonstrated an inverse 150 correlation between AM-8621 sensitivity and BCL-XL expression (19), in agreement with a 151 recent study that assessed the sensitivity of S63845 in a more limited set of hematologic cancer 152 cell lines (18). Comparison of MCL-1 and BCL-XL expression levels in NSCLC and CRC cell 153 lines in the Cancer Cell Line Encyclopedia (CCLE) revealed that as a group, CRC cell lines 154 exhibited higher BCL-XL and lower MCL-1 expression than NSCLC cell lines (Supplemental

155 Figure 3A), consistent with our observation that KRAS mutant CRC cell lines were more 156 sensitive to BCL-XL inhibition (Supplemental Figure 2). However, when examining individual 157 cell lines within our panel of KRAS mutant NSCLC cell lines, we did not observe an obvious 158 correlation between combination drug sensitivity and expression of MCL-1 or BCL-XL mRNA, 159 or ratios of BIM to MCL-1 or BCL-XL (Supplemental Figure 3B). We also did not observe 160 subsets of KRAS mutant NSCLC cell lines with inverse co-expression of BCL-XL and MCL-1 161 (e.g. MCL-1HIGH/BCL-XLLOW or MCL-1LOW/BCL-XLHIGH) that might obviously predict 162 differential drug sensitivity. In general, NSCLC cell lines with greater MCL-1 tended to also 163 have greater BCL-XL expression. A similar pattern was observed in KRAS mutant NSCLC 164 tumors (Supplemental Figure 3C, D). Therefore, baseline expression levels of BCL-XL or MCL- 165 1 may not be sufficiently robust to predict sensitivity to MEK + MCL-1 or BCL-XL inhibition 166 for individual cell lines or tumors. 167 168 To determine the molecular basis for the differential sensitivity to MEK + MCL-1 versus BCL- 169 XL inhibition, we examined the impact of drug treatment on apoptotic signaling. As expected, 170 inhibition of phospho-ERK by trametinib led to accumulation of BIM protein (Supplemental 171 Figure 3E). Treatment with AM-8621 or navitoclax interrupted BIM:MCL-1 and BIM:BCL-XL 172 interactions, respectively (Supplemental Figure 3F), with a commensurate increase in BIM 173 binding to the uninhibited pro-survival partner, consistent with the ability of pro-survival BCL-2 174 family proteins to buffer BIM liberated by BH3 mimetics. To compare the effects of BIM 175 induction on apoptotic priming in H2030 and H1734, we performed BH3 profiling (24) before 176 and after treatment with trametinib. Trametinib treatment increased overall apoptotic priming 177 (BID peptide) in both cell lines, indicating that pro-apoptotic signaling was induced by the 178 treatment (Figure 3A). Notably, in H2030 cells, this corresponded to increased MCL-1 179 dependence, whereas H1734 cells became more BCL-XL dependent, in agreement with the 180 combination treatment data (Figure 2). 181 182 We next examined whether the differences in apoptotic dependency result from differential 183 binding of BIM and other pro-apoptotic BCL-2 proteins to MCL-1 or BCL-XL. At baseline, 184 MCL-1 was localized primarily to the mitochondria in most cell lines, whereas BCL-XL was 185 variably distributed between the mitochondria and cytosol (Supplemental Figure 3G). BIM was

186 exclusively localized to the mitochondrial fraction, indicating that the critical BIM:MCL-1 and 187 BIM:BCL-XL interactions occur at the mitochondria. To directly compare the amount of BIM 188 bound to MCL-1 relative to BCL-XL, we performed immunoprecipitation of MCL-1 and BCL- 189 XL and assessed for binding of pro-apoptotic BCL-2 family members. In A427, H23, and H2030 190 cells, which are MCL-1 dependent, BIM was preferentially bound to MCL-1 at baseline (Figure 191 3B). In contrast, BIM was bound to BCL-XL as well as MCL-1 in BCL-XL dependent H1944 192 and H1734 cells. Additionally, we observed that BAK was bound to MCL-1 in H23 and H2030 193 cells, while BAX was bound to BCL-XL in H1944 and H1734 cells. Treatment of H2030 cells 194 with trametinib led to additional accumulation of BIM at the mitochondria (Figure 3C) and 195 increased binding of BIM to MCL-1, explaining the increase in MCL-1 priming revealed by 196 BH3 profiling (Figure 3D). Treatment with combined trametinib + AM-8621 prevented the 197 accumulation of BIM on MCL-1 and led to a decrease in mitochondrial BIM, suggesting that 198 other pro-survival BCL-2 family proteins such as BCL-XL are insufficient to neutralize and 199 stabilize BIM once it is liberated from MCL-1. In H1734 cells, trametinib treatment led to BIM 200 accumulation on BCL-XL as well as MCL-1; co-treatment with ABT263 + trametinib displaced 201 both BIM and BAX from BCL-XL (Figure 3D). Thus, the sensitivity of H2030 and H1734 cells 202 to combined MEK + BH3 mimetics appears to be determined by a primary dependency on MCL- 203 1 or BCL-XL, respectively, to neutralize multiple pro-apoptotic BCL-2 family members. 204 Together, these results point to the complexity and diversity of interactions between BCL-2 205 family proteins that determine sensitivity to MEK inhibitor + BH3 mimetic combinations. 206 207 Combination MEK + MCL-1 inhibition leads to KRAS mutant NSCLC tumor regression 208 209 To evaluate the in vivo activity of MEK + MCL-1 inhibition, we generated H2030 and A549 210 sub-cutaneous xenograft tumors in immunocompromised mice. Consistent with prior studies of 211 KRAS mutant xenograft tumors (25, 26), treatment with trametinib suppressed phospho-ERK 212 signaling and led to modest tumor growth inhibition in both models (Figure 4A-B, Supplemental 213 Figure 4A-B). To investigate the effects of MCL-1 inhibition in vivo, we used AM-4907, a 214 structural analog of AM-8621 with improved oral bioavailability (Supplemental Figure 1A) (19). 215 AM-4907 modestly slowed the growth of H2030 xenograft tumors, but had little activity against 216 A549 xenograft tumors, largely mirroring the response of the respective cell lines to AM-8621

217 (Supplemental Figure 2B). Combination treatment with trametinib and AM-4907 led to 218 activation of cleaved caspase-3 (Figure 4B) and caused regression of both A549 and H2030 219 xenograft tumors (Figure 4A). Similar results were also observed in A427 xenograft tumors 220 (Supplemental Figure 4C). The differential sensitivity of H2030 xenograft tumors to BH3 221 mimetic combinations was consistent with the in vitro drug sensitivity, with the combination of 222 trametinib + navitoclax showing little activity beyond that of trametinib alone (Supplemental 223 Figure 4D). 224 225 To evaluate the efficacy of MEK + MCL-1 inhibition in additional clinically relevant in vivo 226 models, we generated primary patient-derived xenograft (PDX) mouse tumor models from KRAS 227 mutant adenocarcinomas (Supplemental Table 2) that represented a variety of KRAS point 228 mutations as well as co-occurring mutations. Mice bearing PDX tumors were treated with 229 trametinib or the combination of trametinib + AM-4907 or trametinib + AMG 176 (the clinical 230 analog) for two weeks and evaluated for tumor regression. Whereas tumor regression was 231 observed with single agent trametinib only in 1/8 models (MGH6024, <30%), combination 232 treatment led to average tumor regression in 6/8 models, with four achieving greater than 30% 233 average tumor volume reduction (Figure 4C, Supplemental Figure 4E)). Additionally, for 6/7 234 evaluable models, the combination of trametinib + AM-4907 had statistically greater anti-tumor 235 activity than trametinib alone. In contrast, the combination of trametinib + navitoclax showed 236 minimal activity beyond trametinib alone in 4/4 models tested (Supplemental Figure 4E, F). 237 Consistent with these results, co-immunoprecipitation experiments confirmed that BIM was 238 preferentially bound to MCL-1 in MGH1070 and MGH1089-1 tumors (Supplemental Figure 239 4G). Together, these results demonstrate in vivo efficacy of combined inhibition of MEK and 240 MCL-1 and suggest that this may be a promising therapeutic approach for treatment of KRAS 241 mutant NSCLC in the clinic. 242 243 Prior BCL-XL inhibition increases MCL-1 dependence and increases sensitivity to MCL-1 244 inhibitors 245 246 The development of selective MCL-1 inhibitors with potent in vivo activity now provides the 247 opportunity to rationally design therapies that can specifically target each of the major pro-

248 survival BCL-2 family proteins that protect cells during tyrosine kinase inhibitor treatment (14). 249 However, our results suggest the selection of the most appropriate BH3 mimetic may be 250 hampered by the lack of clinically feasible biomarkers (e.g. gene mutations, RNA or protein 251 expression levels) that can robustly predict whether tumors are MCL-1 or BCL-XL dependent. 252 To circumvent this problem, we investigated whether intermittent treatment with alternating 253 MCL-1 and BCL-XL inhibitors might represent a practical strategy to maximize treatment 254 efficacy without the need to prospectively determine apoptotic dependencies. We treated H2030 255 cells with AM-8621 or navitoclax in combination with trametinib for four days, followed by 256 drug washout for three days (for detailed treatment schemas, see Supplemental Table 3). After 257 washout, cells were re-challenged with either the same or opposite drug combination, for up to 258 three rounds of treatment. Unexpectedly, we observed that after exposure to navitoclax + 259 trametinib, cells became exquisitely sensitive to AM-8621 + trametinib (Supplemental Figure 260 5A). To determine whether "pre-treating" cells with navitoclax might sensitize to MCL-1 261 inhibition, we selected cell lines with varied MCL-1 dependencies and treated them sequentially 262 with navitoclax, drug washout, and finally AM-8621 + trametinib. While navitoclax pre- 263 treatment had little impact on the sensitivity to trametinib alone, cell lines became more sensitive 264 to the AM-8621 + trametinib combination, as indicated by the degree of separation (ΔAUC) 265 between the trametinib only and trametinib + AM-8621 dose-response curves (Figure 5A-C). 266 Conversely, pre-treatment with AM-8621 did not sensitize cells to combined navitoclax + 267 trametinib (Figure 5B,C). The increased sensitivity was observed after a range of navitoclax pre- 268 treatment and drug washout periods (Supplemental Figure 5B) and corresponded with increased 269 apoptosis (Figure 5D). 270 271 We next examined whether navitoclax pre-treatment causes KRAS mutant NSCLC cells to 272 become sensitive to AM-8621 alone. Whereas H2030 cells, which exhibit baseline sensitivity to 273 MCL-1 inhibition, became more sensitive to AM-8621, cell lines with little or no baseline MCL- 274 1 dependence (A549, H1944, H1734) did not become sensitive to single agent AM-8621 275 (Supplemental Figure 5C, D). Thus in the majority of cells, an additional apoptotic stimulus such 276 as BIM induction is still required to trigger an apoptotic response, suggesting that sequential 277 application of BCL-XL followed by MCL-1 inhibitors might selectively sensitize KRAS mutant 278 NSCLC cells to MEK inhibition. To test this, we compared the response of KRAS mutant

279 NSCLC lines with non-KRAS mutant lung adenocarcinoma, squamous cell carcinoma and non- 280 cancer cell lines. Navitoclax pre-treatment increased the sensitivity of the majority of KRAS 281 mutant NSCLC cell lines to AM-8621 + trametinib (Figure 5E), with the exception of KRAS 282 mutant NSCLC cells that were completely insensitive to both MEK + MCL-1 and MEK + BCL- 283 XL inhibition at baseline (H460, H358). Of note, cell lines that were sensitive to MEK + BCL- 284 XL inhibition but not MEK + MCL-1 inhibition at baseline (H1734, H441) became sensitive to 285 MEK + MCL-1 inhibition after navitoclax pre-treatment. We did not observe sensitization of 286 non-KRAS mutant cells including KRAS wild-type lung cancer cells and cancer-associated 287 fibroblast cells, consistent with the notion that this approach selectively targets KRAS mutant 288 cells that have MAPK-mediated suppression of BIM. Finally, to verify that the effect of 289 navitoclax pre-treatment was mediated by BCL-XL, we tested the BCL-XL selective inhibitor 290 A1331852 and observed even more pronounced sensitization in KRAS mutant NSCLC cells, but 291 not in non-KRAS mutant cell lines (Figure 5E). Overall, these results suggest that sequential 292 application of BH3 mimetics can be used to selectively sensitize KRAS mutant NSCLC cells to 293 MEK inhibition. 294 295 To determine the mechanism of increased MEK + MCL-1 inhibitor sensitivity of KRAS mutant 296 cells after transient BCL-XL inhibitor treatment, we first examined whether pretreatment with 297 navitoclax induced changes in the expression of BCL-2 family genes. After both navitoclax 298 treatment and washout, we did not observe any consistent changes in the RNA transcript levels 299 in H2030, H2009, H1734 and H1944 cells that could explain the sensitization (Supplemental 300 Figure 5E) . We next examined the binding interactions and subcellular localization of BCL-2 301 family proteins. In H2030 cells, in which the majority of BIM is already bound to MCL-1 cells at 302 baseline, navitoclax or A1331852 pre-treatment caused a further modest increase in the relative 303 amount of BIM bound to MCL-1 which persisted even after drug washout (Supplemental Figure 304 5F). An even more pronounced increase in BIM bound to MCL-1 was observed in H2009, 305 H1944 and H1734, which exhibit less MCL-1 dependence at baseline but become sensitive to 306 MEK + MCL-1 inhibition after navitoclax or A1331852 pre-treatment (Figure 5F). In contrast, 307 there was little or no increase in BIM bound to MCL-1 after navitoclax or A1331852 pre- 308 treatment in cells that did not sensitize (H358, H460). Conversely, there was no increase in BIM 309 bound to BCL-XL after pretreatment with AM-8621 (Supplemental Figure 5G), consistent with

310 the lack of sensitization to BCL-XL inhibition with the reciprocal pretreatment strategy (Figure 311 5B,C). We also observed additional changes in the sub-cellular localization of BCL-2 family 312 proteins, with an increase in MCL-1 and BIM and/or decrease in BCL-XL protein at the 313 mitochondria (Supplemental Figure 5H, I), all consistent with increased dependence on MCL-1. 314 315 To understand why the effects of navitoclax persist despite drug washout, we quantified the 316 amount of intracellular navitoclax after drug washout. In both H2030 and H1734 cells, 317 approximately 50% of intracellular navitoclax was retained 24 hours after washout, and this 318 remained stable for up to 96 hours (Supplemental Figure 5J). In contrast, AM-8621 was depleted 319 from cells immediately upon removal of drug from the culture media (Supplemental Figure 5K), 320 again consistent with its inability to sensitize cells to BCL-XL inhibition. All together, these 321 results suggest that transient exposure of KRAS mutant NSCLC cells to BCL-XL inhibitors leads 322 to sustained loading of BIM on MCL-1, increasing MCL-1 dependence and thus poising cells to 323 more readily undergo apoptosis upon subsequent treatment with combination MEK + MCL-1 324 inhibitors. Indeed, navitoclax pre-treated H2030 and H1734 cells underwent rapid mitochondrial 325 depolarization and cytochrome C release after treatment with trametinib + AM-8621 compared 326 to vehicle pre-treated cells (Figure 5G, Supplemental Figure 5L). 327 328 Pre-treatment with BCL-XL inhibitors may permit alternative MEK + MCL-1 inhibitor dosing 329 strategies 330 331 The use of MEK inhibitors in the clinic is associated with significant toxicity in many patients 332 (27), and this may limit the promise of MEK-based combination therapies. Alternative drug 333 administration schedules such as intermittent dosing have the potential to minimize toxicity, but 334 may come with the cost of decreased efficacy. We hypothesized that increasing the MCL-1 335 inhibitor sensitivity of KRAS mutant NSCLC cells by pre-treatment with BCL-XL inhibitors 336 might offset decreased activity of intermittent MEK + MCL-1 inhibition. To test this, we 337 monitored cell viability of KRAS mutant NSCLC cell lines during treatment with continuous or 338 intermittent trametinib + AM-8621 (3 days on/4 days off), either after vehicle or navitoclax pre- 339 treatment. We also assessed patient-derived cell lines that corresponded to PDX models used for 340 the in vivo studies (independently generated from the clinical tumor samples, except for

341 MGH1070x which was derived from the established mouse PDX tumor). Intermittent trametinib 342 + AM-8621 was slightly less effective than continuous treatment (P = 0.055) for reducing cell 343 viability (Figure 6A). However, when cells were pre-treated with navitoclax, intermittent 344 trametinib + AM-8621 led to dramatically reduced cell viability in all cell lines. Finally, we 345 tested whether BCL-XL inhibitor pre-treatment sensitizes KRAS mutant xenograft tumors to 346 MEK + MCL-1 inhibition in vivo. H2030 xenografts that were pre-treated with navitoclax or 347 A1331852 experienced deeper and more rapid tumor regressions upon treatment with trametinib 348 + AM-4907 or AMG-176 combinations compared to vehicle pre-treated tumors (Figure 6B). 349 Thus, transient treatment of KRAS mutant NSCLCs with BCL-XL inhibitors increases sensitivity 350 to combined MEK + MCL-1 inhibition in vitro and in vivo, and may enable intermittent dosing 351 strategies with preservation of anti-tumor efficacy.

352 Discussion 353 354 The development of MCL-1 inhibitors with potent and selective in vivo activity represents a 355 major advance in our ability to individually target the three major pro-survival BCL-2 family 356 proteins that regulate the apoptotic response: MCL-1, BCL-2 and BCL-XL. While BH3 mimetics 357 that target these three proteins have demonstrated activity against various hematologic 358 malignancies (18, 19, 28), emerging data suggests that solid tumor malignancies such as NSCLC 359 and breast cancer will be largely refractory to these drugs when used alone. For these cancers, 360 the promise of BH3 mimetics lies in their ability to potentiate the apoptotic response of other 361 therapeutic modalities such as targeted therapies or cytotoxic chemotherapy. 362 363 A number of studies have demonstrated that the BCL-2/BCL-XL inhibitors ABT-737 and ABT- 364 263 (navitoclax) can synergize with targeted therapies such as EGFR inhibitors in NSCLC (29- 365 31) and MEK inhibitors in BRAF melanoma (32) and KRAS mutant cancers (11). Similarly, two 366 recent studies demonstrated that the novel MCL-1 inhibitor S63845 synergizes with targeted 367 therapies and chemotherapy in breast, melanoma and lung cancer models (18, 33). Our 368 combination drug screen similarly identified strong synergistic associations between the novel 369 spiro-macrocyclic MCL-1 inhibitor AM-8621 and a number of clinically relevant compounds 370 that may warrant further investigation. In this study, we focused on synergy between AM-8621 371 and MEK inhibitors in KRAS mutant lung cancers because of the clear association with an 372 oncogenic driver mutation and the lack of effective targeted therapy options for these patients. In 373 a subset of KRAS mutant NSCLC cell lines, combining the MEK inhibitor trametinib with AM- 374 8621 led to robust activation of apoptosis and potent suppression of cell viability. These results 375 reinforce the notion that therapies that target oncogenic dependencies and induce pro-apoptotic 376 BCL-2 proteins (e.g. induction of BIM upon MEK inhibition) drive dependency on BCL-XL or 377 MCL-1 that can be therapeutically exploited with BH3 mimetics. Combining trametinib with the 378 orally bioavailable analog AM-4907 or the clinical compound AMG 176 induced tumor 379 regressions in KRAS mutant NSCLC models in vivo, providing strong rationale for the clinical 380 investigation of this combination for KRAS mutant NSCLC. 381

382 In order to move beyond proof-of-concept studies and define specific strategies for effectively 383 deploying BH3 mimetics in the clinic, a more precise understanding of the specific functional 384 apoptotic dependencies of different cancer subsets is needed. Now that potent and selective 385 inhibitors that target MCL-1, BCL-2 and BCL-XL are available, it is possible to directly 386 compare parallel approaches targeting each of these proteins. In this study, comparing the 387 response of KRAS mutant NSCLC and CRC cell lines to dual MEK + MCL-1 or BCL-XL 388 blockade yielded several important insights. First, we found that CRC cell lines are more 389 uniformly BCL-XL dependent than NSCLC cell lines. This finding is consistent with the 390 observation that CRC cell lines as a group have higher BCL-XL and lower MCL-1 expression 391 levels, which would be expected to result in increased BCL-XL dependency. Whether this results 392 from differences in co-occurring mutations or the influence of cell lineage remains to be 393 determined, however, it suggests that tissue of origin may play an influential role in determining 394 apoptotic dependencies independent of oncogenic driver. 395 396 Second, our results suggest that the specific apoptotic dependencies of NSCLCs may be 397 heterogeneous, with different KRAS mutant NSCLC cell lines being dependent on MCL-1, BCL- 398 XL, both or neither. Developing reliable and robust biomarkers that can predict these apoptotic 399 dependencies and guide selection of BH3 mimetics will be crucial to guiding patient selection in 400 the clinic. Many studies have demonstrated an inverse correlation between the sensitivity of BH3 401 mimetics and the expression level of the non-targeted pro-survival BCL-2 family protein. For 402 instance, high expression levels of MCL-1 correlate with decreased sensitivity to ABT- 403 737/navitoclax (34-36); conversely, sensitivity to S63845 and AM-8621 inversely correlate with 404 BCL-XL expression levels (18, 19, 33). However, it is not clear if these correlations are 405 sufficiently robust to enable MCL-1 and BCL-XL expression levels to be used to predict drug 406 sensitivity of individual tumors within cancer sub-classes. For our KRAS mutant NSCLC cell line 407 panel, we found that expression levels of BCL-2 family members were insufficient for predicting 408 response to combination MEK + MCL-1 versus MEK + BCL-XL inhibition. Co- 409 immunoprecipitation experiments revealed notable differences in the binding interactions 410 between pro-apoptotic BCL-2 proteins and MCL-1 or BCL-XL in cell lines that had similar 411 levels of MCL-1 and BCL-XL expression, and this corresponded to sensitivity to MCL-1 or 412 BCL-XL inhibition. This indicates that gene or protein expression levels alone incompletely

413 describe the functional apoptotic dependencies that determine response to BH3 mimetics. BH3 414 profiling has recently emerged as a promising method for determining the apoptotic priming 415 state of cancer cells (24, 37, 38), and in this study, it differentiated between cells that were 416 sensitive to trametinib + AM-8621 versus trametinib + navitoclax. It remains to be seen whether 417 BH3 profiling can be effectively applied to solid tumor malignancies for which routine 418 diagnostic core biopsy tissue samples are scant. 419 420 Given these challenges, therapeutic strategies that incorporate both BCL-XL and MCL-1 421 inhibitors might obviate the need to define specific apoptotic dependencies. In our combination 422 drug screen, the combination of AM-8621 with ABT-737 or navitoclax was universally potent, 423 with marked synergy observed against every cell line, suggesting that simultaneous 424 administration might be associated with unacceptable toxicity. This motivated us to explore 425 intercalating intermittent AM-8621 or navitoclax in combination with trametinib, a strategy that 426 might also minimize the thrombocytopenia associated with navitoclax. Unexpectedly, we 427 observed that transient exposure to BCL-XL inhibitors caused cells to become more sensitive to 428 trametinib + AM-8621, including cells that exhibited no sensitivity to MCL-1 inhibition at 429 baseline. On a molecular level, pre-treatment with navitoclax or A1331852 caused displacement 430 of BIM from BCL-XL, retrotranslocation of BCL-XL from the mitochondria to the cytosol and 431 in some cases, an increase in mitochondrial MCL-1. The net result of these changes was an 432 increase in the amount of BIM bound to MCL-1 at the mitochondria, poising the cell for rapid 433 mitochondrial depolarization and release of cytochrome C upon treatment with trametinib + AM- 434 8621. In contrast, cell lines that did not sensitize after navitoclax or A1331852 pre-treatment 435 exhibited very little increase in BIM bound to MCL-1. The fact that the changes in BCL-2 family 436 protein interactions persist even after drug washout is likely facilitated by the long intracellular 437 half-life of navitoclax. Although our studies did not directly determine whether navitoclax 438 remains engaged in the BCL-XL binding pocket after washout, we did not observe restoration of 439 BIM:BCL-XL interactions in H1734 cells after washout (Supplemental Figure 5F). Interestingly, 440 BCL-XL retrotranslocation from mitochondria to cytosol has been previously reported to protect 441 cells from apoptosis by helping to recycle mitochondrial BAX back to its inactive cytoplasmic 442 pool, and disruption of this process by ABT-737 increases both BCL-XL retrotranslocation and 443 mitochondrial BAX localization (39). In our models, we did not observe any changes in the

444 cytosolic-mitochondrial distribution of BAX after navitoclax pre-treatment, suggesting that this 445 mechanism does not account for the observed apoptotic sensitization. 446 447 Although this study focused specifically on KRAS mutant NSCLC, the strategy of alternating 448 BH3 mimetics that selectively target MCL-1 and BCL-XL may be relevant to other oncogene- 449 addicted cancers in which BH3-only proteins such as BIM can be induced by targeted therapies 450 (40). It is noteworthy that sensitization after BCL-XL inhibitor pre-treatment only occurred in 451 cell lines that exhibited at least partial sensitivity to either MEK + MCL-1 or MEK + BCL-XL 452 inhibition. KRAS mutant NSCLC cells that were completely insensitive to both trametinib + BH3 453 mimetic combinations did not become more sensitive MEK + MCL-1 inhibitors after exposure to 454 BCL-XL inhibitors, nor did KRAS wild-type lung cancer cells or stromal fibroblasts, which are 455 not driven by oncogenic MAPK pathway activation. This suggests that matching the targeted 456 therapy to oncogenic driver remains a critical determinant of selectivity, with BH3 mimetics 457 acting as a potentiator of the apoptotic response. Thus these results provide a rationale for further 458 investigation of intercalated and intermittent dosing strategies that may maximize the efficacy of 459 BH3 mimetic - targeted therapy combinations in diverse cancer types.

460 Materials and Methods 461 462 Cell lines, antibodies and reagents 463 KRAS mutant NSCLC and CRC cell lines were obtained from the Center for Molecular 464 Therapeutics at the MGH Cancer Center which performs routine SNP and STR authentication. 465 Additionally, cell lines underwent STR validation at the initiation of the project (Biosynthesis, 466 Inc.). Cell lines were routinely tested for mycoplasma during experimental use. Cell lines were 467 maintained in RPMI supplemented with 5% FBS except A427, SW1573, H2009, H1573, GP5d, 468 LS174T, LS1034, LoVo, SW1116, SW837 and T84 cells which were maintained in DMEM/F12 469 supplemented with 5% FBS. Patient-derived NSCLC and cancer-associated fibroblast cell lines 470 were established in our laboratory from surgical resections, core needle biopsies or pleural 471 effusion samples as previously described (41), with the exception of MGH1070x cell line which 472 was derived from a primary mouse PDX model. All patients signed informed consent to 473 participate in a Dana Farber/Harvard Cancer Center Institutional Review Board-approved 474 protocol giving permission for research to be performed on their samples. Clinically observed 475 KRAS mutations (determined by MGH SNaPshot NGS genotyping panel) were verified in 476 established cell lines. Established patient-derived cell lines were maintained in RPMI + 10% 477 FBS. For cell culture and in vivo studies, Trametinib, Navitoclax (ABT-263), Venetoclax (ABT- 478 199), A1331852 were purchased from Selleckchem. AM-8621, AM-4907 and AMG176 were 479 provided by Amgen. For cell culture studies, drugs were dissolved in DMSO to a final 480 concentration of 10 mM and stored at −20°C; unless otherwise specified, 1 μM concentration 481 was used for in vitro cell culture experiments. For western blotting, the following antibodies 482 were used: MCL-1, BCL-XL, BIM, BAK, PUMA, Cytochrome-C, COX-IV, HDAC1, beta- 483 tubulin, pERK1/2 T202/204, ERK1/2, beta-actin, (Cell Signaling); NOXA, BAX (Santa Cruz). 484 485 Combination drug screen 486 Viability Assay for Combination Screen: The AM-8621 combination screen was performed at 487 Zalicus (Cambridge, MA). In brief, cells were seeded in growth media in black 1536-well tissue 488 culture treated plates at optimized seeding densities to ensure log phase growth throughout the 489 compound treatment duration. Cells were incubated at 37°C and 5% CO2 for 24 hours before 490 treatment. At the time of treatment, a set of assay plates (which do not receive treatment) was

491 collected and ATP levels were measured via ATPLite viability assay (Perkin Elmer). These 492 Tzero (T0) plates were read using ultrasensitive luminescence on Envision Plate Readers. 493 Treated assay plates were incubated with compound for 72 hours (see Combination Screen 494 Design below). After 72 hours, plates were developed for endpoint analysis using ATPLite. All 495 data points were collected via automated processes, quality controlled, and analyzed using 496 Chalice software (Zalicus). Assay plates were accepted if they passed the following quality 497 control standards: relative luciferase values were consistent throughout the entire experiment, Z- 498 factor scores were greater than 0.6, untreated/vehicle controls behaved consistently on the plate. 499 Growth Inhibition (GI) values were calculated as a measure of cell viability. The cell viability of 500 vehicle is measured at the time of dosing (T0) and after 72 hours (T72). A GI reading of 0% 501 represents no growth inhibition - cells treated with compound and T72 vehicle signals are 502 equivalent. A GI of 100% represents complete growth inhibition - cells treated with compound 503 and T0 vehicle signals are equivalent. A GI of 200% represents complete death of all cells in the 504 culture well. GI was calculated by applying the following test and equation: 505 506 507 Where T is the signal measure for a test article or combination, V is the vehicle-treated control

508 measure, and V0 is the vehicle control measure at time zero. This formula is derived from the 509 Growth Inhibition calculation used in the National Cancer Institute’s NCI-60 high throughput 510 screen. 511 Combination Screen Design: Combination analysis data was collected in a 7x7 512 checkerboard optimized matrix for 15 cell lines screened in the 1536-well format. 187 enhancer 513 compounds were combined with AM-8621 across the 15 cell line panel. In addition, 40 514 compounds were combined in self-cross analysis for each cell line. The starting concentrations 515 and fold dilution for AM-8621 and enhancer compounds were optimized to best fit the individual 516 single agent dose response curves. 517 Synergy Score Analysis: To measure combination effects in excess of Loewe additivity, a 518 scalar measure was used to characterize the strength of synergistic interaction termed the 519 Synergy Score. The Synergy score is calculated as:

520 The fractional inhibition for each component agent and combination point in the matrix is 521 calculated relative to the median of all vehicle-treated control wells. The Synergy Score equation 522 integrates the experimentally-observed activity volume at each point in the matrix in excess of a 523 model surface numerically derived from the activity of the component agents using the Loewe 524 model for additivity. Additional terms in the Synergy Score equation (above) are used to 525 normalize for various dilution factors used for individual agents and to allow for comparison of 526 synergy scores across an entire experiment. The inclusion of positive inhibition gating or an 527 Idata multiplier removes noise near the zero effect level, and biases results for synergistic 528 interactions that occur at high activity levels. 529 Self-Cross Based Combination Screen Analysis: To objectively establish hit criteria for 530 the AM-8621 combination screen, 40 compounds from the combination library were selected to 531 be self-crossed across the 15 cell line panel as a means to empirically determine a baseline 532 additive, non-synergistic response. The identity of the 40 self-cross compounds was determined 533 by selecting compounds with a variety of maximum response values and single agent dose 534 response steepness. Those drug combinations, which yielded effect levels that statistically 535 superseded those baseline additivity values, were considered synergistic. 536 The Synergy Score measure was used for the self-cross analysis. Synergy Scores of self-crosses 537 are expected to be additive by definition and, therefore, maintain a Synergy Score of zero. 538 However, while some self-cross Synergy Scores are near zero, many are greater suggesting that 539 experimental noise or non-optimal curve fitting of the single agent dose responses are 540 contributing to the slight perturbations in the score. Given the potential differences in cell line 541 sensitivity to AM-8621 combination activities, we used a cell-line centric strategy for the self- 542 cross based combination screen analysis, focusing on self-cross behavior in individual cell lines 543 versus global review of the cell line panel activity. Combinations where the Synergy Score was 544 greater than the mean self-cross plus two standard deviations (2σ) were considered candidate 545 synergies at the 95% confidence levels. A total of 960 hits were identified at 2σ above the mean 546 (32% hit rate). 547 Confirmatory High Resolution Synergy Experiments: For confirmatory synergy studies 548 reported in Figure 1C similar methodologies to those described for the AM-8621 combination 549 screen were employed with the following exceptions: (1) studies were performed in 384 well

550 plates (2) combinations and self-cross experiments were performed in 10 x 10 complete 551 optimized matrices. All experiments were performed in duplicate. 552 553 Cell proliferation and viability assays 554 For cell proliferation dose response assays, cell lines were seeded into 96-well plates 24 hours 555 before addition of drug. Cell proliferation was determined by CellTiter-Glo assay (Promega) 72 556 hours after adding drug according to the manufacturers protocol. For short-term time course 557 experiments, plates were drugged in identical fashion at indicated time points; all plates in an 558 experiment were developed with CellTiter-Glo simultaneously. Cell viability studies were 559 performed using crystal violet staining. Cells were fixed with glutaraldehyde, washed with water 560 and stained with 0.1% crystal violet for 30 minutes. After imaging, staining was quantified by 561 using 10% acetic acid to extract the stain and absorbance read at 590nm. Long-term cell viability 562 studies were performed using RealTime-Glo assay (Promega) according to the manufacturer's 563 protocol. Cell viability was assessed at the indicated time points and fresh media was replaced 564 immediately afterward. For navitoclax pre-treatment studies, we observed similar sensitization 565 over a range of pre-treatment and washout time periods (48-72 hours); for consistency, 48 hour 566 pre-treatment/48 hour washout was used unless otherwise indicated. 567 568 PI/Annexin apoptosis assay 569 Cells were seeded in triplicates at low density 24 hours prior to drug addition. 72 hours after 570 adding drugs, floating and adherent cells were collected and stained with propidium iodide and 571 Cy5-Annexin V (BD Biosciences) and analyzed by flow cytometry. The annexin-positive 572 apoptotic cell fraction was analyzed using FloJo software. 573 574 Caspase 3/7 apoptosis assay 575 Cells were seeded in triplicates at low density 24 hours prior to drug addition. 48 hours after 576 adding drug, caspase cleavage was quantified using ApoTox-Glo Triplex Assay (Promega). 577 Relative caspase cleavage was determined as follows: (luminescence)/[400Ex/505Em 578 (Viability)]. 579 580 Subcellular Fractionation assay

581 Cells were incubated with drug for designated time periods. Following incubations, whole cell, 582 cytosolic, mitochondrial and nuclear fractions were sequentially isolated using a cell 583 fractionation kit (Abcam, catalogue number 109719) according to the manufacturer’s protocol, 584 with minor modifications. Purity of fractions was determined by Western blot analysis using 585 specific nuclear, cytosolic and mitochondrial markers. 586 587 Immunoprecipitation assay 588 Immunoblotting was performed per antibody manufacturer’s specifications. Cells were lysed 589 using Tris Lysis Buffer and Protease inhibitor cocktail (Meso Scale Diagnostics) and 590 immunoprecipitated overnight at 4℃ with Protein A/G Agarose beads (Thermo Fisher) and the 591 following antibodies: anti-MCL-1 (BD Pharmigen); anti-BCL-XL (EMD Millipore); anti-mouse 592 IgG1 isotype control (Cell Signaling). The immunoprecipitate, the supernatant, and a sample of 593 the initial whole cell lysate for each condition were then analyzed by western blot. 594 595 siRNA knockdown studies 596 Cells were plated in Opti-MEM media (Invitrogen), transfected with 50 nM siRNAs specific for 597 MCL-1 (Thermo Fisher, 4392420; Qiagen, S10218120S) using HiPerfect. 24 hours after 598 transfection, cells were seeded for experiments. Cell lysate for western blotting of MCL-1 were 599 collected 48 hours after transfection. For caspase 3/7 activity assays, drugs were added starting 600 48 hours after transfection. 601 602 BH3 profiling assay 603 BH3 profiling was performed as previously described (38). Briefly, cells were treated with 604 trametinib at 100 nM for 16 hours and JC1-based BH3 profiling was performed on untreated 605 versus trametinib-treated cells. Change in apoptotic priming was defined as the difference in 606 extent of mitochondrial depolarization induced by BID, MS1 and HRK peptides in treated versus 607 untreated cells. 608 609 Navitoclax washout studies 610 H2030 and H1734 cells were seeded in 10 cm dishes at a density of 5 x 105 cells/dish in 10 mL 611 growth media containing 5% FBS. Plates were incubated overnight at 37°C and 5% CO2 to

612 allow cells to adhere to the bottom of the plates. Cells were then treated with 1 μM navitoclax or 613 AM-8621 for 72 hours, followed by washout and replacement with 10 mL of compound free 614 growth media. At indicated time points post washout, media was aspirated from plates followed 615 by two washes with 5 mL PBS. Plates were then stored at -80°C. To prepare for liquid 616 chromatography-mass spectrometry (LC-MS) analysis, plates were thawed and 5 mL of 617 acetonitrile was added to each plate followed by incubation at room temperature with shaking for 618 20 minutes. Samples were then spun down to collect supernatant, which was dried down and 619 reconstituted in equal parts acetonitrile and water followed by analysis on an API 5500 mass 620 spectrophotometer. 621 622 Mouse Xenograft Assays and Drug Treatment 623 All mouse studies were conducted through Institutional Animal Care and Use Committee– 624 approved animal protocols in accordance with institutional guidelines. KRAS mutant NSCLC 625 patient-derived xenograft models were generated from surgical resections, core needle biopsies 626 or pleural effusion samples by sub-cutaneous implantation into NSG mice (Jackson Labs). Sub- 627 cutaneous tumors were serially passaged twice to fully establish each model. Clinically observed 628 KRAS mutations were verified in each established model. For drug studies, PDX tumors were 629 directly implanted sub-cutaneously into NSG or athymic nude (NE/Nu) mice and allowed to 630 grow to 250-400 mm3. For H2030 and A549 xenograft studies, cell line suspensions were 631 prepared in 1:1 matrigel:PBS and 5x106 cells were injected unilaterally into the subcutaneous 632 space on the flanks of athymic nude (Nu/Nu) mice and allowed to grow to approximately 350 633 mm3. Tumors were measured with electronic calipers and the tumor volume was calculated 634 according to the formula V = 0.52 x L x W2. Mice with established tumors were randomized to 635 drug treatment groups using covariate-adaptive randomization to minimize differences in 636 baseline tumor volumes. Tumor volumes at the start of treatment (mean +/- S.D.): A549 control - 637 372 +/- 28 mm3, A549 trametinib 382 +/- mm3, A549 AM-4907 374 +/- 22 mm3, A549 638 trametinib+AM-4907 382 +/- 11 mm3; H2030 control 315 +/- 42 mm3, H2030 trametinib 318 +/- 639 32 mm3, H2030 AM-4907 318 +/- 60 mm3, H2030 trametinib+AM-4907 +/- mm3; H2030 640 navitoclax 227 +/- 55 mm3, H2030 trametinib + navitoclax 283 +/- 66 mm3; A427 control 201 641 +/-20 mm3, A427 trametinib 318 +/- 16 mm3, A427 AM-4907 312 +/- 17 mm3, A427 642 trametinib+AM-4907 305 +/- 19 mm3. Trametinib was dissolved in 0.5% HPMC/0.2% Tween

643 80 (pH 8.0) and administered by oral gavage daily 3 mg/kg, 6 days per week. AM-4907 and 644 AMG176 were dissolved in 25% hydroxypropyl-beta-cyclodextrin (pH8.0) and administered by 645 oral gavage daily at 100 mg/kg and 50 mg/kg, respectively. Navitoclax was dissolved in 60% 646 Phosal 50/30% PEG 400/10% ethanol and administered by oral gavage daily 100 mg/kg. 647 A13318752 was dissolved in 60% Phosal 50/27.5% PEG 400/10% ethanol/2.5% DMSO and 648 administered by oral gavage 25 mg/kg twice daily. 649 650 Immunohistochemistry 651 Histology was performed by HistoWiz Inc. (histowiz.com) using standard operating procedures 652 and fully automated workflow. Formalin-fixed paraffin-embedded KRAS mutant NSCLC 653 xenograft tumors were sectioned at 4μm. Immunohistochemistry was performed on a Bond Rx 654 autostainer (Leica Biosystems) with enzyme treatment (1:1000) using standard protocols. 655 Antibodies used were: phospho-ERK (1:100, #4307 Cell Signaling) and cleaved caspase-3 656 (1:300, #9661 Cell Signaling). Bond Polymer Refine Detection (Leica Biosystems) was used 657 according to manufacturer’s protocol. Sections were then counterstained with hematoxylin, 658 dehydrated and film coverslipped using a TissueTek-Prisma and Coverslipper (Sakura). 659 660 Confocal Fluorescence Microscopy 661 Cells were seeded at low density on a Lab-Tek Chamber Slide System 48 hours prior to drug 662 treatment. Cells were treated with 1μM A1331852 or vehicle for 48 hours, washed with PBS, 663 then cultured for 48 hours in media without drug. Cells were incubated with MitoTracker Red 664 CMXRos (ThermoFisher) for 30 minutes, fixed with 1% paraformaldehyde and permeabilization 665 with 0.2% Titron X-100. Cells were then blocked with 5% NGS/5% BSA for 1 hour and 666 incubated with 1:500 anti-BCL-XL rabbit mAb (54H6, Cell Signaling) overnight at 4oC. Cells 667 were washed with 0.05% Tween 20, then incubated with 1:500 anti-rabbit IgG Alexa Fluor 488 668 conjugate (Cell Signaling) for 1 hour at room temperature. Finally cells were stained with 1:1000 669 DAPI for 10 minutes. Slides were imaged the next day on a Zeiss LSM 710 inverted laser 670 scanning confocal microscope (Excitation/Emission: 495/519 nm for BCL-XL, 578/608 nm for 671 MitoTracker) at 63x oil magnification. 672 673 Data and Statistical Analysis

674 Data were analyzed using GraphPad Prism software (GraphPad Software). Unless otherwise 675 specified, data displayed are mean and standard error. Pair-wise comparisons between groups 676 (e.g., experimental versus control) were made using paired or unpaired t-tests as appropriate. For 677 xenograft tumor measurements, individual time points were compared using multiple t tests with 678 Holm-Sidak correction for multiple comparisons. Unless otherwise indicated, P values below 679 0.05 were considered to be statistically significant 680

681 Acknowledgements:

682 We thank members of the Hata Lab for helpful discussions and feedback. This study was funded

683 by support from the Conquer Cancer Foundation of ASCO (A. Hata), Uniting Against Lung

684 Cancer (A. Hata), NIH R01CA14059408 (C. Benes), Lungstrong, and Be a Piece of the Solution.

685 This project was also supported by a Stand Up To Cancer-American Cancer Society Lung

686 Cancer Dream Team Translational Research Grant (SU2C-AACR-DT17-15). Stand Up to

687 Cancer is a division of the Entertainment Industry Foundation. Research grants are administered

688 by the American Association for Cancer Research, the Scientific Partner of SU2C.

689

690 Author Contributions

691 F.M.S., V.N., S.C., P.E.H., and A.N.H. designed the study, analyzed the data and wrote the

692 paper. F.M.S., V.N., S.C., M.G.C., H.L.A, C.L., D.R. and A.N.H. performed cell line and

693 biochemical studies. D.T., S.J.B. N.P. and M.G.C. performed tumor xenograft studies. S.J.B,

694 M.G.C, and A.N.H. generated the patient derived cell lines and mouse PDX models. C.F. and

695 K.A.S performed the BH3 profiling studies. K.V., L.A.F., M.L., C.D.W., K.A.R., D.P.C, J.H.S.,

696 C.K., L.V.S., Z.P., A.F.F., C.G.A., and J.F.G., provided KRAS mutant NSCLC tumor tissue

697 from which cell lines and PDX models were derived. S.B. synthesized MCL-1 inhibitors. V.N.

698 and F.M.S. contributed equally to the study. All authors discussed the results and commented on

699 the manuscript.

700

701 Competing financial interests

702 The authors declare competing financial interests: A.N.H. has provided consulting services for

703 Amgen and receives research support from Amgen, Novartis and Relay Therapeutics. S.C.,

704 D.R., S.B., A.C. and P.E.H. are employees of Amgen, as noted in the affiliations.

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Figure Legends

Figure 1. Synergy between AM-8621 and MAPK pathway inhibitors in cell lines with MAPK pathway activation. A, AM-8621 synergizes with multiple classes of drugs in diverse cancer cell lines. The number of cell lines in the combination drug screen in which statistically significant synergy was observed for the majority of the compounds in a given drug category is shown. The top eight drug classes (which represent 53 of 187 drugs) with the most synergistic interactions are shown. Gray hatched bars represent specific drug sub-class indicated in parentheses (BCL-2 specific inhibitors, MEK inhibitors). Synergy scores for individual compounds and cell lines are shown in Supplemental Figure 1E. B, Synergy scores for AM-8621 combined with MEK and BRAF inhibitors across the cell line panel. Strong synergy is indicated in red. Asterisks denote cell lines with activating mutations in the MAPK pathway. Hash marks indicate cell lines with known functional activation of MAPK signaling. C, Heterologous and self-cross drug combinations confirm synergy between AM-8621 and clinically relevant MEK/BRAF inhibitors in cell lines with oncogenic mutations that lead to MAPK pathway activation.

Figure 2. KRAS mutant NSCLC cell lines exhibit a heterogeneous pattern of synergy between MEK inhibitors and BH3 mimetics. A, Cell lines were treated with trametinib in combination with AM-8621 or navitoclax in a 8x6 dose matrix and synergy was calculated according to the Loewe excess additivity model. B, Cell lines were treated with increasing concentrations of trametinib alone or in the presence of 1 μM AM-8621 or navitoclax for 72 hours and viability was determined. The enhancement of growth inhibition by the addition of AM-8621 or navitoclax to trametinib was determined by normalizing the viability after combination treatment to trametinib alone for each dose point (0 = no effect, 100% = complete cell killing). The lower panel compares the relative enhancement achieved by the addition of AM-8621 (red) or navitoclax (blue) to trametinib for the entire KRAS NSCLC cell line panel. C, Combining trametinib (100 nM) with AM-8621 (1 μM) or navitoclax (1 μM) leads to increased apoptosis as determined by annexin-V staining. D, Effect of combining trametinib (100 nM) with AM-8621 (1 μM) or navitoclax (1 μM) on cell viability. Cells were treated for 72 hours and stained with crystal violet.

Figure 3. Mitochondrial BCL-2 family interactions determine sensitivity profile to BH3 mimetics. A, The change in apoptotic priming after treatment with 100 nM trametinib for 16 hours was determined by BH3 profiling, which assesses mitochondria depolarization (MOMP) after treatment with BH3 peptides. Total apoptotic priming is determined by BID peptide, while MS1 and HRK peptides are specific for MCL-1 and BCL-XL dependency, respectively. Data are the mean and standard error of four independent experiments. B, Direct binding interactions between BCL-2 family proteins. MCL-1 and BCL-XL were immunoprecipitated and interacting proteins were determined by western blotting (Ig – Immunoglobulin control, In – total cell lysate input, IP – immunoprecipitated fraction, S – supernatant). Right panel summarizes binding of pro-apoptotic BCL-2 family proteins to MCL-1 versus BCL-XL. C, Sub-cellular localization of BCL-2 family proteins after 24 hours drug treatment. D, Effect of drug treatment on binding interactions between BCL-2 family proteins. Cells were treated with 100 nM trametinib, 1 μM AM-8621 and/or 1 μM navitoclax as indicated for 24 hours (M – MCL-1 immunoprecipitation, B – BCL-XL immunoprecipitation, In – Input, sm- MCL-1 supernatant, sb – BCL-XL supernatant).

Figure 4. Combined MEK and MCL-1 inhibition leads to tumor regression in vivo. A, The combination of trametinib (3 mg/kg daily) +AM-4907 (100 mg/kg daily) leads to tumor regression of A549 and H2030 sub-cutaneous xenograft tumors. A549: each treatment group N = 7 animals. H2030: each treatment group N=7, except control N=5. Data shown are mean and standard error, with asterisks denoting a statistical significance difference (P < 0.05) between combination treatment and trametinib alone (as determined by multiple t-test with Holm Sidak correction for multiple comparisons). B, Trametinib + AM-4907 treatment causes suppression of phospho-ERK and activation of cleaved caspase-3 in A549 xenograft tumors. Scale bar = 50 mm. Right panel, CC3 positive cells were counted in three high power fields for two independent xenograft tumors for each drug treatment. C, Combined MEK + MCL-1 inhibition leads to tumor regression of KRAS mutant NSCLC PDX tumors. Mice were treated with trametinib (3 mg/kg, diamonds) alone or in combination with AM-4907 (100 mg/kg, solid circles) or AMG 176 (50 mg/kg, open circles). Asterisks indicate a statistically significant (P < 0.05) decrease in

combination treatment compared with trametinib alone. Right panel, number of individual animals treated with each drug.

Figure 5. Transient BCL-XL inhibition alters BCL-2 family interactions and increases sensitivity to MCL-1 inhibition. A, Navitoclax pre-treatment increases sensitivity to combination MEK + MCL-1 inhibition. Cells were pre-treated with navitoclax for 48 hours followed by drug washout for 48 hours, then treated with trametinib +/- 1 μM AM-8621 for 72 hours (blue boxes = navitoclax, red boxes = AMG-8621, open boxes = vehicle, line = drug washout period). For detailed treatment schemas, see Supplemental Table 3. B, Navitoclax pre- treatment increases sensitivity to combined MEK + MCL-1 inhibition, however the reciprocal strategy of AM-8621 pre-treatment does not increase sensitivity to MEK + BCL-XL inhibition. ΔAUC corresponds to shaded area between trametinib only (black) and trametinib + BH3 mimetic (colored) curves and is a measure of the sensitization effect. C, Comparison of the increase in sensitization observed with the reciprocal pre-treatment strategies. Data are replotted from Panel B, and each dot represents the increase in MCL-1 sensitization after pre-treatment with navitoclax (red) or BCL-XL sensitization after pre-treatment with AM-8621 (blue) for each cell line. D, Cells were pre-treated with 1 μM navitoclax for 48 hours followed by drug washout for 48 hours, then treated with 100 nM trametinib, 1 μM navitoclax, 1 μM AM-8621 or combination for 72 hours. Apoptosis was determined by PI/annexin staining. E, Navitoclax or A1331852 pre-treatment sensitizes KRAS mutant NSCLC cells that exhibit baseline sensitivity to either MEK + MCL-1 or MEK + BCL-XL inhibition, but not KRAS mutant NSCLC cells without baseline sensitivity or non-KRAS mutant cells. F. Navitoclax or A1331852 pre-treatment leads to increased BIM bound to MCL-1 in cells that exhibit sensitization (H2009, H1944, H1734), but not in cells that do not sensitize (H358, H460). G, Navitoclax pre-treated cells undergo rapid mitochondrial depolarization and release of cytochrome C upon treatment with trametinib + AM-8621.

Figure 6. Pre-treatment BCL-XL inhibitors increases efficacy of combined MEK + MCL-1 inhibition. A, KRAS mutant NSCLC cell lines were treated with vehicle (“A”), continuous trametinib + AM-8621 (“B”), intermittent (3 days on/4 days off) trametinib + AM-8621 (“C”) or navitoclax followed by intermittent trametinib + AM-8621 (“D”) and cell viability determined at

indicated timepoints in replicate plates. Data are normalized to the cell viability at the time of initial trametinib + AM-8621 treatment. Right panel shows aggregate data for the final time point. B, Mice bearing H2030 sub-cutaneous xenograft tumors were pre-treated with navitoclax (100 mg/kg daily) or A1331852 (25 mg/kg twice daily) for 4 days. After 3 days of drug holiday, mice were treated with trametinib (3 mg/kg) plus AM-4907 (100 mg/kg daily) or AMG 176 (50 mg/kg daily) for five days. Change in tumor volume at the end of two weeks relative to tumor size at the start of MEK + MCL-1 inhibitor treatment is shown.

Exploiting MCL-1 dependency with combination MEK + MCL-1 inhibitors leads to induction of apoptosis and tumor regression in KRAS mutant non-small cell lung cancer

Varuna Nangia, Faria M Siddiqui, Sean Caenepeel, et al.

Cancer Discov Published OnlineFirst September 25, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-18-0277

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