MCL-1 dependency as a novel vulnerability for aggressive B cell lymphomas

Dear Editor, Mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL) are aggressive hematologic malignancies characterized by the accumulation of lymphoid cells defective in cell apoptosis biology and function. The anti-apoptotic B cell lymphoma 2 (BCL-2) family proteins are pivotal regulators of the mitochondrial apoptotic pathway and genetic aberrations in these genes are associated with lymphomagenesis and chemotherapeutic resistance. These anti-apoptotic proteins, most notably BCL-2 and myeloid cell leukemia 1 (MCL-1), promote the survival of lymphoma cells by counteracting the activity of pro-apoptotic proteins such as BCL-2-like protein 11 (BCL2L11, also known as BIM). Pharmacological targeting of BCL-2 is highly effective in certain B cell lymphomas, but de novo and acquired resistance to BCL-2 inhibitor monotherapy often develop, especially in aggressive B cell lymphomas. Moreover, the other major anti-apoptotic BCL-2 family proteins, BCL-XL and MCL1, are demonstrated to be the main determinants of resistance to venetoclax. Additionally, MCL-1 is recurrently highly expressed in various kinds of non-Hodgkin’s B cell lymphomas. Collectively, these data support the hypothesis that MCL-1 plays a central role in B cell lymphoma progression and drug resistance. Pharmacologically targeting MCL-1, therefore, represents an attractive strategy to combat these lymphomas. To this end, there is a great need to develop and apply selective small-molecule MCL-1 inhibitors as part of a first-line lymphoma therapy or upon emergence of tumor resistance characterized by upregulation of MCL-1. Here, we exploited the MCL-1 dependency in MCL and DLBCL by implementing pharmacogenomic and chemical proteomic approaches to investigate the molecular drug response and resistance mechanism to MCL-1 inhibitors. We demonstrated that transcriptome and kinome reprograming linked to the MEK and ERK pathways contribute to MCL-1 inhibitor resistance via regulation of the BCL-2 family profile. Further analysis revealed synergistic activity of MCL-1 inhibitors in combinations with inhibitors of MEK, ERK, and BCR in MCL-1 inhibitor-resistant MCL/ DLBCL lines and primary samples. These results provide a strong rationale for further evaluation of MCL-1 inhibitor in combination with established therapy in the clinical setting and highlight a potential strategy for overcoming MCL-1 inhibitor resistance. First, we affirmed the dependency of MCL-1 in MCL and DLBCL survival and growth. We performed cellviability assays in a panel of MCL and DLBCL lines for their susceptibility to specific MCL-1 inhibitor, S63845. As shown in Fig. 1A, most MCL and DLBCL lines were highly sensitive to S63845. Additionally, MCL-1 inhibition with S63845 triggered dramatic PARP cleavage in MCL/ DLBCL lines and primary samples, indicating that the compound’s inhibitory effect was attributed to induced mitochondrial-mediated apoptosis (Fig. S1A). These results are in line with the notion that MCL and DLBCL typically have high expression of MCL-1 as well as BCL-2 (refs. ). While targeting MCL-1 appears to be a viable therapeutic strategy, previous clinical and pre-clinical data suggest that treatment with single-agent anti-BCL-2 family member therapy is associated with the rapid acquisition of resistance. In anticipation of the evolution of MCL-1 inhibitor resistance, we developed MCL-1 resistance (MR) models


MCL-1 dependency as a novel vulnerability for aggressive B cell lymphomas
Michelle Y. Wang 1 , Tao Li 1 , Yuan Ren 1 , Bijal D. Shah 2 , Tint Lwin 1 , Jing Gao 1 , Kenneth H. Shain 1,2,3 , Wei Zhang 4 , Xiaohong Zhao 1 and Jianguo Tao 1,5 Dear Editor, Mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL) are aggressive hematologic malignancies characterized by the accumulation of lymphoid cells defective in cell apoptosis biology and function 1 . The anti-apoptotic B cell lymphoma 2 (BCL-2) family proteins are pivotal regulators of the mitochondrial apoptotic pathway and genetic aberrations in these genes are associated with lymphomagenesis and chemotherapeutic resistance. These anti-apoptotic proteins, most notably BCL-2 and myeloid cell leukemia 1 (MCL-1), promote the survival of lymphoma cells by counteracting the activity of pro-apoptotic proteins such as BCL-2-like protein 11 (BCL2L11, also known as BIM) [2][3][4] . Pharmacological targeting of BCL-2 is highly effective in certain B cell lymphomas, but de novo and acquired resistance to BCL-2 inhibitor monotherapy often develop, especially in aggressive B cell lymphomas. Moreover, the other major anti-apoptotic BCL-2 family proteins, BCL-XL and MCL-1, are demonstrated to be the main determinants of resistance to venetoclax 5 . Additionally, MCL-1 is recurrently highly expressed in various kinds of non-Hodgkin's B cell lymphomas 6 . Collectively, these data support the hypothesis that MCL-1 plays a central role in B cell lymphoma progression and drug resistance. Pharmacologically targeting MCL-1, therefore, represents an attractive strategy to combat these lymphomas. To this end, there is a great need to develop and apply selective small-molecule MCL-1 inhibitors as part of a first-line lymphoma therapy or upon emergence of tumor resistance characterized by upregulation of MCL-1.
Here, we exploited the MCL-1 dependency in MCL and DLBCL by implementing pharmacogenomic and chemical proteomic approaches 5,7 to investigate the molecular drug response and resistance mechanism to MCL-1 inhibitors. We demonstrated that transcriptome and kinome reprograming linked to the MEK and ERK pathways contribute to MCL-1 inhibitor resistance via regulation of the BCL-2 family profile. Further analysis revealed synergistic activity of MCL-1 inhibitors in combinations with inhibitors of MEK, ERK, and BCR in MCL-1 inhibitor-resistant MCL/ DLBCL lines and primary samples. These results provide a strong rationale for further evaluation of MCL-1 inhibitor in combination with established therapy in the clinical setting and highlight a potential strategy for overcoming MCL-1 inhibitor resistance.
First, we affirmed the dependency of MCL-1 in MCL and DLBCL survival and growth. We performed cellviability assays in a panel of MCL and DLBCL lines for their susceptibility to specific MCL-1 inhibitor, S63845. As shown in Fig. 1A, most MCL and DLBCL lines were highly sensitive to S63845. Additionally, MCL-1 inhibition with S63845 triggered dramatic PARP cleavage in MCL/ DLBCL lines and primary samples, indicating that the compound's inhibitory effect was attributed to induced mitochondrial-mediated apoptosis (Fig. S1A). These results are in line with the notion that MCL and DLBCL typically have high expression of MCL-1 as well as BCL-2 (refs. 6,8 ). While targeting MCL-1 appears to be a viable therapeutic strategy 9 , previous clinical and pre-clinical data suggest that treatment with single-agent anti-BCL-2 family member therapy is associated with the rapid acquisition of resistance.
In anticipation of the evolution of MCL-1 inhibitor resistance, we developed MCL-1 resistance (MR) models by treating S63845-sensitive cell lines with high doses of S63845 for an extended period in MCL, DLBCL, and MCL-derived lines (10 × IC 50 for 3 months). Though parental lines were exquisitely sensitive to S63845, the IC 50 s for S63845 were more than 10-100-fold higher in all derived resistant lines (Fig. S1B). Similarly, these MR lines showed a comparable level of resistance to the chemically distinct MCL-1 inhibitor, AZD5991 (Fig. S1C).   1C and S1F). However, the decrease in ABT-199 sensitivity was not as dramatic as the increase in S63845 sensitivity, which we attributed to the remaining expression of BCL-2 as well as the strong potency and selectivity of ABT-199. To further confirm the importance of BCL-2 in MCL-1 inhibitor resistance, we tested the effect of ABT-199 in MR lines and found that similar to BCL-2 KD, BCL-2 inhibition enhanced the potency of S63845 in these cells (Fig. S1G). Together, these data indicated that altered dependency on BCL-2 contributed to S63845 resistance. Next, we performed RNA-sequencing on parental and resistant SUDHL-16 cells. Analysis of the transcriptomes of the sensitive and resistant cells revealed a set of differentially expressed genes in the resistant phenotype, with 359 upregulated genes and 194 downregulated genes in MR (Fig. S1H). Implementation of gene set enrichment analysis (GSEA) revealed a significant positive enrichment of MEK-MAPK, mTOR, and BCR pathways in SUDHL-16 MR cells (Fig. 1D). Chemical activity-based proteomic profiling 7,10 was performed in parallel on paired SUDHL-16-sensitive and -resistant cells. Consistent with our previous findings 5, 10 , the gene expression programs identified by RNA-seq translated to kinome reprogramming in SUDHL-16 MR cells with the activation of many kinase pathways, including the BTK, MEK, and ERK pathways (Fig. 1E). Increased MEK/ERK signaling was confirmed by western blot, illustrating increased ERK phosphorylation and BIM downregulation in S63845resistant lines (SUDHL-16, HBL-2, Mino, Marver-1, Fig. 1F). Collectively, these data demonstrated that transcriptome and kinome reprogramming in MR lymphoma cells were associated with upregulation of the MEK-ERK, AKT-mTOR, and BCR pathways, which may converge on BCL-2 and BIM to promote S63845 resistance.
To determine the functional role of transcriptome and kinome reprogramming in BCL-2 family protein expression and S63845 resistance, a drug sensitivity screen composed of 31 small-molecule kinase inhibitors 5,7,10 was performed on paired parental and MR cells (SUDHL-16, Mino, HBL-2). Additionally, we used S63845 as an "anchor" to determine the key signaling inhibitors that reinstated S63845 sensitivity in resistant cells. In line with our transcriptome and kinome results, MR cells exhibited higher sensitivity to inhibitors of the MEK (Trametinib), ERK (SCH772984), and BCR pathways (Ibrutinib, R406, Fig. S2A). Importantly, the combination of S63845 with these inhibitors showed enhanced potencies against MR lines ( Fig. 2A). Cell viability and colony formation assays validated synergistic effects of the combination of S63845 with Trametinib or SCH772984 in all tested MR lines (Fig. S2B, C). Similar enhanced effects of these combination treatments were observed in MCL and DLBCL cell lines with pre-existing MCL-1 inhibitor resistance (Figs. 1A and S2D), indicating that this is an effective approach to combat both de novo and acquired resistance to MCL-1 inhibition.
Next, we capitalized on a novel ex vivo microfluidic cellbased platform 5,7,10 to assess the response of a panel of kinase inhibitors as well as the combination of S63845 with these kinase inhibitors in a 3D reconstructed tumor microenvironment on primary MCL samples collected from lymph node and peripheral blood 11,12 . Again, we   observed enhanced effects of S63845 combined with Trametinib or SCH772984 and synergistic effects of both combinations in S63845-resistant primary MCL samples (Fig. 2B, C). Mechanistically, western blot analysis showed that both MEK and ERK inhibition induced BCL-2 downregulation and BIM upregulation in MR lines (SUDHL-16). Furthermore, the combination of S63845 with Trametinib or SCH772984 triggered increased PARP cleavage compared to S63845, MEK, or ERK inhibitor alone in SUDHL-16 and Mino-resistant models (Figs. 2D and S2E). These data indicate that kinome reprograming linked to the MEK and ERK pathways contributed to MCL-1 inhibitor resistance via regulation of the BCL-2 family profile (BCL-2, BIM), and represent a novel vulnerability in MCL-1 inhibitor-resistant lymphoma.
Finally, we integrated RNA-seq data with drug response profiling data for 24 primary MCL samples. We determined drug sensitivity by calculating the area under curve (AUC) and the half maximal effective concentration (EC 50 ) of each drug for every patient. As shown in Fig. 2E, using the AUC of S63845, we segregated these primary samples into responders (n = 10) and non-responders (n = 14) and correlated S63845 response with the gene expression profile of each sample. Consistent with our in vitro results, the investigation of differentially expressed genes of these two response groups by GSEA revealed enrichment of mTOR, MEK-ERK, and BCR pathwayregulated genes in S63845-resistant primary samples (Figs. 2F and S2F). Collectively, these data identify a targetable MEK-ERK dependent regulation of MCL-1 inhibitor resistance with significant translational potential in future clinical combinations trials.
In conclusion, we identified MCL-1 as a key determinant of cell survival and growth in MCL and DLBCL lymphomas, which conferred exquisite sensitivity to MCL-1 inhibition. Using pharmacogenomics and chemical proteomics, we identified that MEK-ERK and BCR pathway activation drive MCL-1 inhibitor resistance in these lymphomas via transcriptional upregulation of BCL-2 (refs. 13,14 ) and post-translational downregulation of BIM 15 . To this end, combinatorial therapies of MCL-1 inhibitor with MEK, ERK, or BCR inhibitors (to decrease BCL-2 and increase BIM) exhibited potent synergistic effects against MCL-1 inhibitor-resistant lymphoma ex vivo and in vivo. Together, these studies provide a rationale for MCL-1 inhibition in MCL/DLBCL and present MCL-1 inhibition-based combination strategies to MCL-1-dependent and MCL-1 inhibitor-resistant B cell lymphomas.