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Mantle cell lymphoma (MCL) is a rare, incurable hematological malignancy with a heterogenous presentation and clinical course. A wide variety of chemotherapy-based regimens are currently utilized in untreated patients. Several targeted or small molecule therapies have shown efficacy in the relapsed/refractory (R/R) setting over the last several years and have since been explored in the frontline setting. Lenalidomide plus rituximab was explored in a phase II study of 38 untreated patients with MCL ineligible for transplant, where the combination produced durable remissions. We looked to build upon this regimen by adding venetoclax to the combination. We conducted a multi-center, open-label, non-randomized, single arm study to evaluate this combination. We enrolled 28 unselected patients with untreated disease irrespective of age, fitness or risk factors. Lenalidomide was dosed at 20 mg daily on days 1-21 of each 28-day cycle. The dose of venetoclax was determined using the TITE-CRM model. Rituximab was dosed at 375 mg/m2 weekly starting on cycle 1 day 1 until cycle 2 day 1. No DLTs were noted. All patients were treated with venetoclax at the maximum tolerated dose of 400 mg daily. The most common Adverse Events noted were neutropenia and thrombocytopenia. The overall and complete response rates were 96% and 86% respectively. 86% of patients achieved minimal residual disease undetectability by NGS. The median overall and progression-free survivals were not reached. The combination of lenalidomide, rituximab and venetoclax is a safe and effective regimen in patients with untreated MCL. Clinical trial # NCT03523975.

PDF - 3347KB, Synthesis and characterization of the lead compound 1 (UMI-59) and its analog 2 (UMI-77), additional details for used methods, and nine supplementary figures.

Supplementary Table S1 shows 17-AAG IC50 in 6 CRC cell lines with different KRAS, BRAF, PIK3CA, p53 and FBW7 genotypes. Supplementary Fig. S1 shows induction of proapoptotic Bcl-2 family proteins by 17-AAG in FBW7-mutant colon cancer cells. Supplementary Fig. S2 shows that FBW7 is critical for cell death and Mcl-1 degradation induced by Hsp90 inhibitors in DLD1 and SW48 cells. Supplementary Fig. S3 shows that Mcl-1 depletion induced by Hsp90 inhibitors is mediated by protein degradation, but not decreased mRNA expression. Supplementary Fig S4 shows that necroptosis and autophagy induced by 5-fluorouracil is not affected in Mcl-1-KI cells.

Using a high-throughput screening (HTS) approach, we have identified and validated several small-molecule Mcl-1 inhibitors (SMI). Here, we describe a novel selective Mcl-1 SMI inhibitor, 2 (UMI-77), developed by structure-based chemical modifications of the lead compound 1 (UMI-59). We have characterized the binding of UMI-77 to Mcl-1 by using complementary biochemical, biophysical, and computational methods and determined its antitumor activity against a panel of pancreatic cancer cells and an in vivo xenograft model. UMI-77 binds to the BH3-binding groove of Mcl-1 with Ki of 490 nmol/L, showing selectivity over other members of the antiapoptotic Bcl-2 family. UMI-77 inhibits cell growth and induces apoptosis in pancreatic cancer cells in a time- and dose-dependent manner, accompanied by cytochrome c release and caspase-3 activation. Coimmunoprecipitation experiments revealed that UMI-77 blocks the heterodimerization of Mcl-1/Bax and Mcl-1/Bak in cells, thus antagonizing the Mcl-1 function. The Bax/Bak-dependent induction of apoptosis was further confirmed using murine embryonic fibroblasts that are Bax- and Bak-deficient. In an in vivo BxPC-3 xenograft model, UMI-77 effectively inhibited tumor growth. Western blot analysis in tumor remnants revealed enhancement of proapoptotic markers and significant decrease of survivin. Collectively, these promising findings show the therapeutic potential of Mcl-1 inhibitors against pancreatic cancer and warrant further preclinical investigations. Mol Cancer Ther; 13(3); 565–75. ©2013 AACR.

Table S1. Sequences of PCR primers used for vector construction and PCR screening of Mcl-1-KI cell lines, and for analysis of Mcl-1 mRNA expression.

Figure S1. HDACi promote GSK3β-mediated Mcl-1 phosphorylation, but not Mcl-1 degradation, in colon cancer cells. SAHA or MS-275 treatment did not affect Mcl-1 protein half-life in HCT116 cells, or induce Mcl-1 degradation in DLD1 and RKO cells. The 4A (S121A/E125A/S159A/T163A) mutant was more effective than WT Mcl-1 in suppressing SAHA- and MS-275-induced apoptosis in DLD1 and RKO cells. Figure S2. Knock-in of the 4A (S121A/E125A/S159A/T163A) mutant Mcl-1 suppressed apoptosis induced by HDACi and other anticancer agents. Figure S3. FBW7 was induced by the kinase inhibitors regorafenib and sorafenib, but not by the HDACi SAHA and MS-275 in colon cancer cells. Transfection of WT but not mutant FBW7 was sufficient to promote Mcl-1 degradation in cells treated with SAHA or MS-275. Figure S4. PUMA, Bim and Noxa mRNA was induced in HCT116 cells in response to SAHA and MS-275 treatment. Bim and Noxa were knocked out by homologous recombination in HCT116 cells. Figure S5. SAHA- and MS-275-induced apoptosis in HCT116 cells was suppressed by knockout of PUMA, Bim, Noxa or BAX. Figure S6. Immunoprecipitation analysis showed that the mutant Mcl-1 in the Mcl-1-KI cells had enhanced binding to Bim and Noxa in response to SAHA or MS-275 treatment. Figure S7. Mcl-1 inhibitors restored SAHA- and MS-275-induced apoptosis analyzed by annexin V/PI staining in Mcl-1-KI cells.

MDM2 oncoprotein binds directly to the p53 tumor suppressor and inhibits its function in cancers retaining wild-type p53. Blocking this interaction using small molecules is a promising approach to reactivate p53 function and is being pursued as a new anticancer strategy. The spiro-oxindole MI-43, a small-molecule inhibitor of the MDM2-p53 interaction, was designed and examined for its cellular mechanism of action and therapeutic potential in colon cancer. MI-43 binds to MDM2 protein with a Ki value of 18 nmol/L and is 300 times more potent than a native p53 peptide. MI-43 blocks the intracellular MDM2-p53 interaction and induces p53 accumulation in both normal and cancer cells, with wild-type p53 without causing p53 phosphorylation. Induction of p53 leads to modulation of the expression of p53 target genes, including up-regulation of p21 and MDM2 in normal primary human cells and in colon cancer cells with wild-type p53. Using HCT-116 isogenic colon cancer cell lines differing only in p53 status or RNA interference to knockdown expression of p53 in the RKO colon cancer cell line, we show that the cell growth inhibition and cell death induction by MI-43 is p53 dependent. Furthermore, induction of cell cycle arrest by MI-43 is dependent on p53 and p21. In normal cells, MI-43 induces cell cycle arrest but not apoptosis. This study suggests that p53 activation by a potent and specific spiro-oxindole MDM2 antagonist may represent a promising therapeutic strategy for the treatment of colon cancer and should be further evaluated in vivo and in the clinic. [Mol Cancer Ther 2008;7(6):1533–42]

Figure S1. Regorafenib and sorafenib did not affect Mcl-1 mRNA expression but induced GSK3β-dependent Mcl-1 degradation. A transfected quadruple mutant in 4 putative phosphorylation sites (S121A/E125A/S159A/T163A) of Mcl-1 was resistant to regorafenib-induced ubiquitination and degradation. Figure S2. Apoptosis induced by regorafenib or sorafenib was suppressed in HCT116 cells with Mcl-1 phosphorylation site mutant knock-in (Mcl-1-KI), inhibition of GSK3β, or knockout of BAX. Figure S3. Mcl-1 degradation and PUMA induction did not affect each other in regorafenib-induced apoptosis. Regorafenib-induced apoptosis in Mcl-1-KI cells could not be restored by the Bcl-2/Bcl-XL inhibitors ABT-737 or ABT-263.

The Bcl-2 family protein Mcl-1 is often degraded in cancer cells subjected to effective therapeutic treatment, and defective Mcl-1 degradation has been associated with intrinsic and acquired drug resistance. However, a causal relationship between Mcl-1 degradation and anticancer drug responses has not been directly established, especially in solid tumor cells where Mcl-1 inhibition alone is insufficient to trigger cell death. In this study, we present evidence that Mcl-1 participates directly in determining effective therapeutic responses in colon cancer cells. In this setting, Mcl-1 degradation was induced by a variety of multikinase inhibitor drugs, where it relied upon GSK3β phosphorylation and FBW7-dependent ubiquitination. Specific blockade by genetic knock-in (KI) abolished apoptotic responses and conferred resistance to kinase inhibitors. Mcl-1-KI also suppressed the antiangiogenic and anti-hypoxic effects of kinase inhibitors in the tumor microenvironment. Interestingly, these same inhibitors also induced the BH3-only Bcl-2 family protein PUMA, which is required for apoptosis. Degradation-resistant Mcl-1 bound and sequestered PUMA from other prosurvival proteins to maintain cell survival, which was abolished by small-molecule Mcl-1 inhibitors. Our findings establish a pivotal role for Mcl-1 degradation in the response of colon cancer cells to targeted therapeutics, and they provide a useful rational platform to develop Mcl-1–targeting agents that can overcome drug resistance. Cancer Res; 77(9); 2512–21. ©2017 AACR.

Supplementary Material from Reactivation of p53 by a specific MDM2 antagonist (MI-43) leads to p21-mediated cell cycle arrest and selective cell death in colon cancer

Mcl-1, a prosurvival Bcl-2 family protein, is frequently overexpressed in cancer cells and plays a critical role in therapeutic resistance. It is well known that anticancer agents induce phosphorylation of Mcl-1, which promotes its binding to E3 ubiquitin ligases and subsequent proteasomal degradation and apoptosis. However, other functions of Mcl-1 phosphorylation in cancer cell death have not been well characterized. In this study, we show in colon cancer cells that histone deacetylase inhibitors (HDACi) induce GSK3β-dependent Mcl-1 phosphorylation, but not degradation or downregulation. The in vitro and in vivo anticancer effects of HDACi were dependent on Mcl-1 phosphorylation and were blocked by genetic knock-in of a Mcl-1 phosphorylation site mutant. Phosphorylation-dead Mcl-1 maintained cell survival by binding and sequestering BH3-only Bcl-2 family proteins PUMA, Bim, and Noxa, which were upregulated and necessary for apoptosis induction by HDACi. Resistance to HDACi mediated by phosphorylation-dead Mcl-1 was reversed by small-molecule Mcl-1 inhibitors that liberated BH3-only proteins. These results demonstrate a critical role of Mcl-1 phosphorylation in mediating HDACi sensitivity through a novel and degradation-independent mechanism. These results provide new mechanistic insights on how Mcl-1 maintains cancer cell survival and suggest that Mcl-1–targeting agents are broadly useful for overcoming therapeutic resistance in cancer cells.Significance: These findings present a novel degradation–independent function of Mcl-1 phosphorylation in anticancer therapy that could be useful for developing new Mcl-1–targeting agents to overcome therapeutic resistance. Cancer Res; 78(16); 4704–15. ©2018 AACR.

PDF file, 90K, Co-localization of CCN6 and BMP4 in clinical samples of breast cancer.

PDF file, 12K, CCN6 overexpression significantly decreased tumor growth in vivo.

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PDF file, 98K, List of TGF-B superfamily genes affected by CCN6 knockdown in HME cells measured by real time PCR.

PDF file, 121K, Confocal images and immunoblot analysis of HME CCN6 KD and control cells grown in 3D for 15 days. CCN6 KD led to disruption of acinar organization and decreased expression of E-cadherin protein. β-Actin was used as loading control.

Supplementary Methods, Figures 1-15 from SM-164: A Novel, Bivalent Smac Mimetic That Induces Apoptosis and Tumor Regression by Concurrent Removal of the Blockade of cIAP-1/2 and XIAP

PDF file, 330K, A. rhCCN6 protein rescues the upregulation of P-p38 and the invasive phenotype due to CCN6 KD in HME cells. B. p38 kinase activity inhibitor SB203580 reversed the invasive phenotype of CCN6 KD HME cells. C. Inhibition of p38 kinase by 20 �M SB203850 or 10 �M SB202190 is sufficient to reduce invasion of SUM149 IBC cell line and aggressive MDA-MB-231 breast cancer cells.

Supplementary Figures 1-5 from A Novel BH3 Mimetic Reveals a Mitogen-Activated Protein Kinase–Dependent Mechanism of Melanoma Cell Death Controlled by p53 and Reactive Oxygen Species

PDF file, 100K, A. Effect of CCN6 KD on P-Smad1, P-Smad5 and total proteins. CCN6 KD increased P-Smad1 while it downregulated P-Smad5. B. Immunoblot showing effective Smad1 siRNA inhibition in HME CCN6 KD and controls. β-Actin was used as loading control. Invasion assays show that Smad1 siRNA has not significant effect on invasion of CCN6 KD or control cells.

The RAS/BRAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is emerging as a key modulator of melanoma initiation and progression. However, a variety of clinical studies indicate that inhibiting the MAPK pathway is insufficient per se to effectively kill melanoma cells. Here, we report on a genetic and pharmacologic approach to identify survival factors responsible for the resistance of melanoma cells to MEK/ERK antagonists. In addition, we describe a new tumor cell–selective means to bypass this resistance in vitro and in vivo. By generating a panel of isogenic cell lines with specific defects in the apoptotic machinery, we found that the ability of melanoma cells to survive in the absence of functional MEK relies on an ERK-independent expression of the antiapoptotic factor Mcl-1 (and to a lesser extent, Bcl-xL and Bcl-2). Using computer-based modeling, we developed a novel Bcl-2 homology domain 3 (BH3) mimetic. This compound, named TW-37, is the first rationally designed small molecule with high affinity for Mcl-1, Bcl-xL, and Bcl-2. Mechanistic analyses of the mode of action of TW-37 showed a synergistic tumor cell killing in the presence of MEK inhibitors. Importantly, TW-37 unveiled an unexpected role of the MAPK pathway in the control of reactive oxygen species (ROS). This function was critical to prevent the activation of proapoptotic functions of p53 in melanoma cells, but surprisingly, it was dispensable for normal melanocytes. Our results suggest that this MAPK-dependent ROS/p53 feedback loop is a point of vulnerability of melanoma cells that can be exploited for rational drug design. (Cancer Res 2006; 66(23): 11348-59)

Supplementary Materials, Methods and Figure Legends 1-5 from A Novel BH3 Mimetic Reveals a Mitogen-Activated Protein Kinase–Dependent Mechanism of Melanoma Cell Death Controlled by p53 and Reactive Oxygen Species

Chromosome 11q23 translocations are present in ~10% of acute leukemia, which generate the oncogenic MLL fusion (MLL-r hereinafter) proteins and drive a subset of aggressive leukemia. Mechanistic studies of MLL-r leukemias implicated several complexes involved in RNA polymerase II-mediated transcription: the Super Elongation Complex (SEC), the DOT1L Complex (DotCom) and the Polymerase Associated Factor 1 Complex (PAF1c). These protein complexes are dysregulated in MLL-r leukemias and amplify transcription of pro-leukemic target genes. The proteins ENL and AF9 are two common MLL fusion partners and share high homology within their N-terminal YEATS domains that function as epigenetic reader domains. Recently, the importance of the wild type ENL (but not AF9) and its epigenetic reader function has been demonstrated in acute leukemias. However, the importance of the YEATS domain in the context of MLL-ENL fusions has not been explored. In patients, we found that most MLL-ENL fusions (84.1%; N=302 patients), but not MLL-AF9 fusions, retain the YEATS domain. These findings prompted us to investigate 1) how the YEATS domain contributes to MLL-ENL leukemogenesis, 2) whether the YEATS domain affects MLL-ENL fusion protein functions, and 3) if YEATS domain presence in MLL-ENL fusion exposes a vulnerability to YEATS inhibitors. Using published YEATS epigenetic reader mutations, we found that the YEATS epigenetic reader function significantly contributes to MLL-ENL leukemogenesis. Disrupting the YEATS epigenetic reader function in MLL-ENL fusion proteins significantly impacts leukemic stem cell frequency. Using an MLL-ENL construct relevant in patients (ΔYEATS hereinafter), we discovered a subset of MLL-ENL targets with altered expression. GSEA revealed several gene signatures enriched in ΔYEATS cells, most interestingly genes downregulated in leukemic stem cells. Specifically, the MLL-ENL target Eya1 is severely disrupted in MLL-ENL YEATS epigenetic reader mutants and ΔYEATS cells. Our mechanistic data suggest that while MLL-ENL binding at Eya1 is impacted in ΔYEATS, YEATS epigenetic reader mutants do not significantly alter MLL-ENL and PAF1c localization. However, YEATS epigenetic reader mutations severely impact epigenetic modifications associated with active transcription, including H3K4me3, H3K9ac and H3K79me2 at the Eya1 locus. Finally, we tested the YEATS inhibitor sensitivity in AML cell lines. We found that the cell line HB1119, which is driven by MLL-ENL fusion with an intact YEATS domain, is among the most sensitive lines to the YEATS inhibitor SGC-iMLLT. Together, our study provides the biological and mechanistic characterizations of the YEATS domain in MLL-ENL leukemias and contributes to the theoretical framework for YEATS inhibitor development in the majority of MLL-ENL patients. Citation Format: Hsiangyu Hu, Nirmalya Saha, Yuting Yang, Ejaz Ahmad, Lauren Lachowski, Uttar Shrestha, Vidhya Premkumar, James P. Ropa, Lili Chen, Blaine Teahan, Sierrah Grigsby, Rolf Marschalek, Zaneta Nikolovska-Coleska, Andrew G. Muntean. The ENL YEATS domain links leukemic stem cell frequency and enhances YEATS inhibitor sensitivity in MLL-ENL leukemias. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3550.

Anti-apoptotic Bcl-2 family proteins are overexpressed in a wide spectrum of cancers and have become well validated therapeutic targets. Cancer cells display survival dependence on individual or subsets of anti-apoptotic proteins that could be effectively targeted by multimodal inhibitors. We designed a 2,5-substituted benzoic acid scaffold that displayed equipotent binding to Mcl-1 and Bfl-1. Structure based design was guided by several solved co-crystal structures with Mcl-1, leading to the development of compound 24, which binds both Mcl-1 and Bfl-1 with K(i) values of 100 nM and shows appreciable selectivity over Bcl-2/Bcl-xL. The selective binding profile of 24 was translated to on-target cellular activity in model lymphoma cell lines. These studies lay a foundation for developing more advanced dual Mcl-1/Bfl-1 inhibitors that have potential to provide greater single agent efficacy and broader coverage to combat resistance in several types of cancer than selective Mcl-1 inhibitors alone.

The task of predicting the interactions between drugs and targets plays a key role in the process of drug discovery. There is a need to develop novel and efficient prediction approaches in order to avoid costly and laborious yet not-always-deterministic experiments to determine drug–target interactions (DTIs) by experiments alone. These approaches should be capable of identifying the potential DTIs in a timely manner. In this article, we describe the data required for the task of DTI prediction followed by a comprehensive catalog consisting of machine learning methods and databases, which have been proposed and utilized to predict DTIs. The advantages and disadvantages of each set of methods are also briefly discussed. Lastly, the challenges one may face in prediction of DTI using machine learning approaches are highlighted and we conclude by shedding some lights on important future research directions.

MLL (KMT2a) translocations are found in ~10% of acute leukemia patients, giving rise to oncogenic MLL-fusion proteins. A common MLL translocation partner is ENL and associated with a poor prognosis in t(11;19) patients. ENL contains a highly conserved N-terminal YEATS domain that binds acetylated histones and interacts with the PAF1c, an epigenetic regulator protein complex essential for MLL-fusion leukemogenesis. Recently, wild-type ENL, and specifically the YEATS domain, was shown to be essential for leukemic cell growth. However, the inclusion and importance of the YEATS domain in MLL-ENL-mediated leukemogenesis remains unexplored. We found the YEATS domain is retained in 84.1% of MLL-ENL patients and crucial for MLL-ENL-mediated leukemogenesis in mouse models. Mechanistically, deletion of the YEATS domain impaired MLL-ENL fusion protein binding and decreased expression of pro-leukemic genes like Eya1 and Meis1. Point mutations that disrupt YEATS domain binding to acetylated histones decreased stem cell frequency and increased MLL-ENL-mediated leukemia latency. Therapeutically, YEATS containing MLL-ENL leukemic cells display increased sensitivity to the YEATS inhibitor SGC-iMLLT compared to control AML cells. Our results demonstrate that the YEATS domain is important for MLL-ENL fusion protein-mediated leukemogenesis and exposes an "Achilles heel" that may be therapeutically targeted for treating t(11;19) patients.

MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. More than 90 different MLL translocation partners have been identified with the most common fusion partners, AF9, ENL, AF4, and AF10. AF9 and ENL are closely related members of the YEATS domain protein family and have been identified as part of several reported complexes including Super Elongation Complex (SEC) and DotCom Complex. AF9 and ENL, through their C-terminal hydrophobic domain, known as ANC1 Homology Domain (AHD), recruit the histone H3K79 methyltransferase hDOT1L to the MLL target genes, constitutively activating transcription of pro-leukemic targets. We have characterized the protein−protein interactions between hDOT1L and MLL fusion oncogenic proteins, AF9 and ENL, on the biochemical, biophysical, and functional level. This study mapped a 10 amino-acid region (aa 865− 874), highly conserved in hDOT1L from a variety of species, as an interacting site with the AHD domain of AF9/ENL proteins and demonstrated that these PPIs are essential for cellular immortalization mediated by MLL-AF9. Applying genetic approach and studying the effects of blocking the AF9-hDOT1L PPIs or hDOT1L enzymatic activity, it was demonstrated complete disruption of hDOT1L recruitment to critical target genes, Hoxa9 and Meis1, in cells expressing hDOT1L mutants with an impaired AF9 binding site. This was followed by significant decrease in H3K79me2 in the promoter regions of these genes, cell differentiation and induction of apoptosis. To identify the potential therapeutic benefits of this novel targeting approach, the effect of these different hDOT1L mutants was investigated on adult murine hematopoiesis. The in vivo studies demonstrated that genetic interventions that result in loss of hDOT1L enzymatic activity rapidly depletes hematopoietic stem and progenitor cells. In contrast, blocking the AF9-hDOT1L PPIs allowed for sustained hematopoiesis. These studies provided an important proof of concept for the potential therapeutic advantage of inhibiting the AF9-hDOT1L interaction and disrupting the integrity of the MLL-fusion complexes. In addition, these results demonstrated that hDOT1L recruitment can be abolished by a single point mutant (I867A), highlighting the crucial role of the hydrophobic interactions and giving insights for further drug discovery. We have reported the first peptidomimetics targeting PPIs between MLL-AF9 and hDOT1L, providing evidence for the druggability assessment of this novel potential therapeutic approach. The AF9/ENL C-terminal domain, is an intrinsically disordered protein, representing a challenge for structural studies. Applying X-ray crystallography, we have successfully determined the 3D structure of the AF9 AHD domain in complex with peptidomimetics establishing a platform for structure-based design of novel, optimized, potent inhibitors, the current focus of our research efforts. Citation Format: Zaneta Nikolovska-Coleska. Characterization of the protein-protein interactions involved in hDOT1L epigenetic regulation on biochemical, structural and functional level towards developing new therapeutic intervention. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA016.

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieve a durable remission. Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that regulates metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we showed that SIRT5 was required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 was required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf Pten–driven melanoma model. Via metabolite and transcriptomic analyses, we found that SIRT5 was required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably included MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a druggable genotype-independent addiction in melanoma.

FBXW7, a classic tumor suppressor, is a substrate recognition subunit of the Skp1-cullin-F-box (SCF) ubiquitin ligase that targets oncoproteins for ubiquitination and degradation. We recently found that FBXW7 is recruited to DNA damage sites to facilitate nonhomologous end-joining (NHEJ). The detailed underlying molecular mechanism, however, remains elusive. Here we report that the WD40 domain of FBXW7, which is responsible for substrate binding and frequently mutated in human cancers, binds to poly(ADP-ribose) (PAR) immediately following DNA damage and mediates rapid recruitment of FBXW7 to DNA damage sites, whereas ATM-mediated FBXW7 phosphorylation promotes its retention at DNA damage sites. Cancer-associated arginine mutations in the WD40 domain (R465H, R479Q and R505C) abolish both FBXW7 interaction with PAR and recruitment to DNA damage sites, causing inhibition of XRCC4 polyubiquitination and NHEJ. Furthermore, inhibition or silencing of poly(ADP-ribose) polymerase 1 (PARP1) inhibits PAR-mediated recruitment of FBXW7 to the DNA damage sites. Taken together, our study demonstrates that the WD40 domain of FBXW7 is a novel PAR-binding motif that facilitates early recruitment of FBXW7 to DNA damage sites for subsequent NHEJ repair. Abrogation of this ability seen in cancer-derived FBXW7 mutations provides a molecular mechanism for defective DNA repair, eventually leading to genome instability.

MLL1 (KMT2A) translocations are found in ~10% of acute leukemia and give rise to an aggressive form of leukemia in infant, pediatric and adult patients. MLL1 fusion driven acute leukemia is characterized by deregulated activity of the Super Elongation Complex (SEC) and the H3K79 methyltransferase DOT1L, which alter the epigenetic landscape and transcription of pro-leukemic MLL1 fusion targets like HoxA9 and Meis1. The Eleven-Nineteen-Leukemia gene (ENL or MLLT1) is a common MLL1 fusion partner and a SEC component. The ENL protein contains a highly conserved N-terminal epigenetic reader YEATS domain that recognizes acetylated H3K9/K18/K27 (H3Kac hereinafter). Wild type ENL was recently found to be essential for leukemic cell growth, which is dependent on its YEATS domain interaction with H3Kac. While this finding highlighted the YEATS domain importance in wild type ENL function in leukemic cells, the inclusion and importance of the YEATS domain in MLL-ENL fusion protein remain to be elucidated. Here, we investigated the clinical relevance and importance of the ENL YEATS domain in MLL-ENL leukemias. We analyzed >300 t(11;19) MLL-ENL leukemia patients for the breakpoint location within the ENL gene and found that the YEATS domain is retained in the resultant MLL-ENL fusion protein in 84.1% of t(11;19) leukemia patients. We tested the importance of the YEATS domain in MLL-ENL mouse models and found that the YEATS domain and downstream sequence is required for MLL-ENL leukemogenesis in vivo. YEATS deletion decreased expression of pro-leukemic targets such as Meis1, an important factor for leukemic stem cells (LSC). To interrogate the contribution of the YEATS epigenetic reader function in MLL-ENL leukemogenesis, we introduced YEATS point mutations rendering the domain defective in interacting with H3Kac and found that this significantly increased leukemia latency in vivo. Further investigation revealed that YEATS point mutations disrupting H3Kac binding significantly decreased MLL-ENL LSC frequency while not affecting homing to the bone marrow. We attribute this LSC frequency change to altered Meis1 expression. Additionally, disruption of the YEATS epigenetic reader function in MLL-ENL leukemia cells does not induce differentiation, apoptosis nor cell cycle arrest. Therapeutically, we predicted the YEATS domain in MLL-ENL would sensitize MLL-ENL leukemia to YEATS domain inhibitors. Indeed, MLL-ENL leukemia cells are more sensitive to the ENL/AF9 YEATS domain inhibitor, SGC-iMLLT, compared to acute leukemia cells driven by other fusions. Together, our results demonstrate that YEATS-H3Kac binding plays an important role in MLL-ENL fusion mediated leukemogenesis. Our data establishes a strong rationale for future exploration of small molecules aimed at disrupting the YEATS-H3Kac interaction as a targeted therapeutics for treating t(11;19) leukemia patients. Citation Format: Hsiangyu Hu, Nirmalya Saha, Ejaz Ahmad, Yuting Yang, Lili Chen, Lauren Lachowski, Blaine Teahan, Sierrah Grigsby, Rolf Marschalek, Zaneta Nikolovska-Coleska, Andrew G. Muntean. The epigenetic reader function of the YEATS domain in MLL-ENL fusion critically affects leukemic stem cell frequency in MLL-ENL leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2966.

Simple Summary MLL-rearranged leukemia, driven by MLL-fusion proteins, is an aggressive, therapy-resistant leukemia found in >60% of infant leukemia and ~10% of adult leukemia. Studies have shown that inhibiting DOT1L enzymatic activity blocks leukemogenesis. However, DOT1L is critical for various normal cellular functions, including hematopoiesis. This study aimed to show that targeting the interaction between the MLL-AF9 fusion and DOT1L would inhibit leukemogenesis while sparing non-leukemic hematopoiesis. We found that disrupting the AF9-DOT1L interaction with a single point mutation was sufficient to impair leukemogenesis. We also demonstrate that genetic interventions that result in loss of DOT1L enzymatic activity in non-leukemic cells rapidly depletes hematopoietic stem and progenitor cells within 7–10 days; however, hematopoiesis was preserved when the AF9-DOT1L interaction was disrupted, leaving the enzymatic function intact. These studies are a proof of concept demonstrating the potential therapeutic advantage of inhibiting the AF9-DOT1L interaction and disrupting the integrity of the MLL-fusion complex. Abstract MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute Dot1l inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9–DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.

Recent advances in drug screening and development strategies have accelerated the discovery of novel anti-cancer modalities. Targeting protein–protein interactions in particular has become a hot area in drug discovery and has enabled the ability to drug prominent therapeutic targets, such as the Bcl-2 family of apoptosis regulators. The Bcl-2 protein family member, Mcl-1, has emerged as a critical target across a vast array of cancers, playing a key role in preventing apoptosis. Many types of cancer rely on Mcl-1 for survival, which promotes de novo and acquired resistance to standard-of-care therapies. Herein, we survey the history and advancement of small molecule Mcl-1 inhibitors, from novel screening approaches and hit optimization to clinical candidates. The drug discovery efforts spanning academia and industry have afforded five different Mcl-1 inhibitors that are currently being evaluated in phase I clinical trials to treat a variety of hematological malignancies. The most forefront and seminal Mcl-1 inhibitor development programs are summarized from their medicinal chemistry efforts and structure activity relationship studies to their biological efficacy demonstrating utility as anti-cancer agents. This chapter serves as a current state of Mcl-1 inhibitor development and contains a wealth of medicinal chemistry knowledge towards drugging protein–protein interactions.

High-throughput screening identifies small molecules that promote cellular stress resistance., Aging is the dominant risk factor for most chronic diseases. Development of antiaging interventions offers the promise of preventing many such illnesses simultaneously. Cellular stress resistance is an evolutionarily conserved feature of longevity. Here, we identify compounds that induced resistance to the superoxide generator paraquat (PQ), the heavy metal cadmium (Cd), and the DNA alkylator methyl methanesulfonate (MMS). Some rescue compounds conferred resistance to a single stressor, while others provoked multiplex resistance. Induction of stress resistance in fibroblasts was predictive of longevity extension in a published large-scale longevity screen in Caenorhabditis elegans, although not in testing performed in worms and flies with a more restricted set of compounds. Transcriptomic analysis and genetic studies implicated Nrf2/SKN-1 signaling in stress resistance provided by two protective compounds, cardamonin and AEG 3482. Small molecules identified in this work may represent attractive tools to elucidate mechanisms of stress resistance in mammalian cells.

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieves a durable remission. SIRT5 is a member of the sirtuin family of protein deacylases that regulate metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we show that SIRT5 is required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 is required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf;Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we find that SIRT5 is required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably include MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a novel, druggable genotype-independent addiction in melanoma.

Mcl-1, a prosurvival Bcl-2 family protein, is frequently overexpressed in cancer cells and plays a critical role in therapeutic resistance. It is well known that anticancer agents induce phosphorylation of Mcl-1, which promotes its binding to E3 ubiquitin ligases and subsequent proteasomal degradation and apoptosis. However, other functions of Mcl-1 phosphorylation in cancer cell death have not been well characterized. In this study, we show in colon cancer cells that histone deacetylase inhibitors (HDACi) induce GSK3β-dependent Mcl-1 phosphorylation, but not degradation or downregulation. The in vitro and in vivo anticancer effects of HDACi were dependent on Mcl-1 phosphorylation and were blocked by genetic knock-in of a Mcl-1 phosphorylation site mutant. Phosphorylation-dead Mcl-1 maintained cell survival by binding and sequestering BH3-only Bcl-2 family proteins PUMA, Bim, and Noxa, which were upregulated and necessary for apoptosis induction by HDACi. Resistance to HDACi mediated by phosphorylation-dead Mcl-1 was reversed by small-molecule Mcl-1 inhibitors that liberated BH3-only proteins. These results demonstrate a critical role of Mcl-1 phosphorylation in mediating HDACi sensitivity through a novel and degradation-independent mechanism. These results provide new mechanistic insights on how Mcl-1 maintains cancer cell survival and suggest that Mcl-1–targeting agents are broadly useful for overcoming therapeutic resistance in cancer cells. Significance: These findings present a novel degradation–independent function of Mcl-1 phosphorylation in anticancer therapy that could be useful for developing new Mcl-1–targeting agents to overcome therapeutic resistance. Cancer Res; 78(16); 4704–15. ©2018 AACR.

Overexpression of EZH2 in estrogen receptor negative (ER-) breast cancer promotes metastasis. EZH2 has been mainly studied as the catalytic component of the Polycomb Repressive Complex 2 (PRC2) that mediates gene repression by trimethylating histone H3 at lysine 27 (H3K27me3). However, how EZH2 drives metastasis despite the low H3K27me3 levels observed in ER- breast cancer is unknown. Here we show that in human invasive carcinomas and distant metastases, cytoplasmic EZH2 phosphorylated at T367 is significantly associated with ER- disease and low H3K27me3 levels. p38-mediated EZH2 phosphorylation at T367 promotes EZH2 cytoplasmic localization and potentiates EZH2 binding to vinculin and other cytoskeletal regulators of cell migration and invasion. Ectopic expression of a phospho-deficient T367A-EZH2 mutant is sufficient to inhibit EZH2 cytoplasmic expression, disrupt binding to cytoskeletal regulators, and reduce EZH2-mediated adhesion, migration, invasion, and development of spontaneous metastasis. These results point to a PRC2-independent non-canonical mechanism of EZH2 pro-metastatic function., Polycomb group protein EZH2 is overexpressed in ER- breast cancer, promoting metastasis. Here, the authors show that independent of the polycomb group, phosphorylation of EZH2 at T367 by p38 promotes cytoplasmic localization of EZH2, binding to vinculin and other regulators of cell migration and invasion.

Heat shock protein 90 (Hsp90) is widely overexpressed in cancer cells and necessary for maintenance of malignant phenotypes. Hsp90 inhibition induces tumor cell death through degradation of its client oncoproteins and has shown promises in preclinical studies. However, the mechanism by which Hsp90 inhibitors kill tumor cells is not well-understood. Biomarkers associated with differential sensitivity and resistance to Hsp90 inhibitors remain to be identified. In this study, we found that colorectal cancer cells containing inactivating mutations of FBW7, a tumor suppressor and E3 ubiquitin ligase, are intrinsically insensitive to Hsp90 inhibitors. The insensitive colorectal cancer cells lack degradation of Mcl-1, a prosurvival Bcl-2 family protein. Hsp90 inhibition promotes GSK3β-dependent phosphorylation of Mcl-1, which subsequently binds to FBW7 and undergoes ubiquitination and proteasomal degradation. Specifically blocking Mcl-1 phosphorylation by genetic knock-in abrogates its degradation and renders in vitro and in vivo resistance to Hsp90 inhibitors, which can be overcame by Mcl-1–selective small-molecule inhibitors. Collectively, our findings demonstrate a key role of GSK3β/FBW7-dependent Mcl-1 degradation in killing of colorectal cancer cells by Hsp90 inhibitors and suggest FBW7 mutational status as a biomarker for Hsp90-targeted therapy. Mol Cancer Ther; 16(9); 1979–88. ©2017 AACR.

The Pax gene family encodes DNA binding transcription factors that control critical steps in embryonic development and differentiation of specific cell lineages. Often, Pax proteins are re-expressed or ectopically expressed in cancer and other diseases of abnormal proliferation, making them attractive targets for tissue specific inhibition by small molecules. In this report, we used a homology model of the Pax2 paired domain and a virtual screen to identify small molecules that can inhibit binding of the paired domain to DNA and Pax2 mediated transcription activation. Candidates from the virtual screen were then confirmed in a cell based Pax2 transactivation assay. Subsequently, we tested analogs of these hits to identify a single compound that effectively blocked Pax2 activity and DNA binding with a Kd of 1.35–1.5 µM. The compound, termed EG1, was used to inhibit embryonic kidney development, a process directly dependent on Pax2 activity. Furthermore, we show that EG1 can inhibit proliferation of Pax2 positive renal and ovarian cancer cell lines but has little effect on Pax2 negative cancer cells. These data confirm that small molecules targeting the DNA binding paired domain can be identified and may be good lead compounds for developing tissue and cell-type specific anticancer therapies.

Approximately 10% of acute leukemia involves rearrangement at chromosome 11q23, giving rise to a relatively aggressive form of acute leukemia characterized by MLL1 (KMT2A) fusion proteins. Despite the identification of >100 MLL1 fusion partners, the majority are members of several similar transcriptional activation complexes including: The Super Elongation Complex (SEC), AEP and EAP (SEC used hereafter). MLL fusion-driven acute leukemia is characterized by deregulated activity of the SEC and the H3K79 methyltransferase DOT1L. This leads to altered epigenetic landscapes at and deregulated transcription of pro-leukemic MLL1-fusion target genes like HoxA9 and Meis1. Thus, targeting these transcriptional and epigenetic complexes has become an attractive therapeutic strategy for treating MLL-fusion leukemia. Eleven-Nineteen-Leukemia (ENL or MLLT1) is the third most common MLL1 fusion partner and a component of the SEC. Recently, wild type ENL was identified as an essential factor for leukemic cell growth. The ENL protein possesses a C-terminal ANC-homology domain (AHD) necessary for SEC recruitment and is essential for MLL-fusion mediated leukemogenesis. In addition, ENL contains a highly conserved N-terminal YEATS domain that functions as an epigenetic reader for acetylated H3K9, H3K18 or H3K27, which is essential for leukemic cell growth. Additionally, the ENL YEATS domain directly interacts with the Polymerase Associated Factor 1 complex (PAF1c), an epigenetic regulator protein complex essential for MLL-fusion mediated leukemogenesis. These studies highlight the importance of the YEATS domain in regulating wild type ENL function in leukemic cells. However, the importance of the YEATS domain in the context of MLL-ENL mediated leukemia remains to be elucidated. In this study, we investigate the clinical relevance and leukemic importance of the ENL YEATS domain in MLL-ENL leukemias. We first analyzed t(11;19) (MLL-ENL) patient data to determine the sites of chromosomal translocation within the ENL gene. We found that the YEATS domain (coded by exons 2 through 4) is retained in 84.1% of MLL-ENL patients (n=302). Specifically, 50.7% (n=153) of these patients possess breakpoints located 5' of the first exon of the ENL gene, while 33.4% (n=101) of the patients display breakpoints within the first intron of ENL gene. These data point towards a tendency for YEATS domain retention in MLL-ENL fusion proteins in t(11;19) patients. We next tested whether the YEATS domain was functional in MLL-ENL mouse leukemia models. Our data shows the YEATS domain is required for MLL-ENL leukemogenesis in vivo, as deletion of the YEATS domain destroys MLL-ENL leukemogenesis and increases apoptosis in cell culture. Transcriptionally, deletion of the YEATS domain decreased expression of pro-leukemic genes such as Meis1 and the anti-apoptotic gene Bclxl. To dissect the contribution of different YEATS domain functions in MLL-ENL leukemogenesis, we engineered YEATS domain mutants defective in interacting with PAF1 or acetylated H3K9/K18/K27. Disrupting the YEATS-PAF1 or YEATS-H3Kac interaction decreased MLL-ENL mediated colony formation exvivo and significantly increased leukemia latency in vivo. The MLL-ENL YEATS domain mutants will be used in future studies to determine how the YEATS domain affects 1) MLL-ENL fusion localization, 2) key protein complexes localization (i.e. SEC and PAF1c) and 3) the epigenetic landscapes (i.e. H3K79me2/3 and H3K4me3) at pro-leukemic targets. To further interrogate the YEATS-PAF1 interaction in MLL-ENL mediated leukemia, we identified the minimal region of the PAF1 protein required for the YEATS-PAF1 interaction. This PAF1 protein fragment will be used to biochemically characterize the structure of the PAF1-YEATS interaction, which might aid in therapeutically targeting specific YEATS interactions in MLL-ENL leukemia. Our results demonstrate for the first time, to our knowledge, an essential role for the YEATS domain in MLL-ENL mediated leukemogenesis. Additionally, our genetic studies elucidate the importance of the YEATS domain interaction with either the PAF1c or H3Kac in MLL-ENL leukemias. Taken together, our study establishes a rationale for exploring the effectiveness of small molecule development aimed at disrupting either the YEATS-H3Kac or the YEATS-PAF1 interaction as a therapeutic intervention for treating MLL-ENL leukemia patients. Disclosures No relevant conflicts of interest to declare.

Context Novel treatment approaches for patients with secondary acute myeloid leukemia (AML) are urgently needed. Secondary AML constitutes about 25-30% of AML and it has been observed that patients with secondary AML have worse responses rates to chemotherapy, increased rates of relapse, and worse overall survival compared to patients with de novo AML. Objective Our long-term goal is to develop more effective therapeutic options for patients with secondary AML. Our objectives in this study are: (i) to demonstrate synergy between cytarabine and venetoclax in secondary AML cell lines and patient-derived samples, (ii) to elucidate the mechanism of this synergy, (iii) functional characterization of Bcl-2 family proteins by BH3 profiling performed on secondary AML cell lines and patient samples. Results Synergy was demonstrated between cytarabine and venetoclax in secondary AML and de novo AML cell lines (Molm13, MV4;11, SKM-1; ML-2) using both an MTT cell viability assay and a flow-cytometry based Annexin-V assay, quantified by the computer software Compusyn. BH3 profiling of these cell lines indicated a dependence on both Bcl-2 and Mcl-1. Interestingly, SKM-1 showed sole dependence on Mcl-1. In SKM-1 cells, western blotting showed increased expression of Mcl-1 after treatment with venetoclax for 8 hrs and decreasing Mcl-1 expression with time (8hrs–72hrs) after treatment with cytarabine. Combination treatment for 24-48 hrs led to Mcl-1 expression that was similar to or less than Mcl-1 expression in control cells. Synergy between cytarabine and venetoclax was confirmed in patient samples. Importantly, BH3 profiling showed that patient samples exhibited dependence on Bcl-2 or both Mcl-1 and Bcl-2. Conclusions There is significant synergy between cytarabine and venetoclax in both de novo and secondary AML cell lines as well as patient samples. One of the potential mechanisms of this synergy is abrogation of Mcl-1 upregulation. Secondary AML exhibits variable functional dependence on Mcl-1 and Bcl-2. These findings support the clinical investigation of cytarabine and venetoclax in patients with secondary AML as well as the use of BH3 profiling as a potential predictive biomarker. Acknowledgements This work was supported by the NIH R01 CA-217141 and AACR Bayer Innovation and Discovery Award to Z. Nikolovska-Coleska.

Biological aging is the dominant risk factor for most chronic diseases. Development of anti-aging interventions offers the promise of preventing many such illnesses simultaneously. Cellular stress resistance is an evolutionarily conserved feature of longevity. Here, we identify compounds that induced resistance to the superoxide generator paraquat (PQ), the heavy metal cadmium (Cd), and the DNA alkylator methyl methanesulfonate (MMS). Some rescue compounds conferred resistance to a single stressor, while others provoked multiplex resistance. Induction of stress resistance in fibroblasts was predictive of longevity extension in a published large-scale longevity screen in C. elegans. Transcriptomic analysis implicated Nrf2 signaling in stress resistance provided by two protective compounds, cardamonin and AEG 3482. Molecules that conferred stress resistance also induced cellular inflammatory pathways, and other core pathways such as AMPK signaling. Small molecules identified in this work may represent attractive candidates to evaluate for their potential pro-health and pro-longevity effects in mammals.

Anti-apoptotic Bcl-2 family proteins are frequently overexpressed in various cancers and are established therapeutic targets. Cancer cells can either display dependence on individual or subsets of these pro-survival proteins, which gives therapeutic relevance to both selective and multimodal inhibitors. Selective Bcl-2, Bcl-xL, and Mcl-1 inhibitors are being evaluated clinically in a wide spectrum of cancers. Bfl-1 is another homologous anti-apoptotic protein with closest structural relation to Mcl-1, but drug discovery efforts on this protein have been limited to peptide inhibitors. There is increasing evidence of Bfl-1 being a viable therapeutic target, particularly in the context of drug resistance. Both Mcl-1 and Bfl-1 share the selective endogenous binding partner, Noxa, which highlights the relevance of these proteins being therapeutically co-inhibited, especially in cancers where they are commonly overexpressed together. Mcl-1 and Bfl-1 have emerged as key players in melanoma, associated with poor clinical responses to BRAF/MEK pathway inhibitors and cell death resistance, thus representing attractive new therapeutic targets.We report herein the structure-based design, synthesis, SAR, and biological characterization of dual inhibitors displaying equipotent binding to Mcl-1 and Bfl-1. This class of inhibitors was designed based on the validated hit molecule identified in our recently reported integrated high throughput and virtual screening study. Several co-crystal structures of these molecules were solved, which guided the structure-based design efforts and led to the optimization of compounds that bind both Mcl-1 and Bfl-1 with Ki values in the 100 nM range and >250-fold selectivity over Bcl-2/Bcl-xL. Direct binding of optimized inhibitors to the Mcl-1 and Bfl-1 proteins was validated by several biophysical methods, including HSQC-NMR, and bio-layer interferometry (BLI). Selectivity over Bcl-2/Bcl-xL was demonstrated on the cellular level by the ability to selectively bind endogenous Mcl-1 and Bfl-1 and disrupt interactions with Bim. On-target cellular activity was further confirmed using Eµ-Myc lymphoma cell lines, which stably overexpress individual anti-apoptotic Bcl-2 proteins with a strong survival dependence on each of these targets. Eµ-Myc cells overexpressing Mcl-1 and Bfl-1 showed dose-dependent cell death in response to treatment with the most potent compounds, while cells overexpressing Bcl-2 and Bcl-xL were not affected even at the highest tested concentration, further demonstrating the selective targeting of Mcl-1 and Bfl-1. With a selective set of chemical tools, we employed the BH3 profiling assay across a panel of melanoma cell lines in order to functionally dissect survival dependence. The obtained data suggests that melanoma cell lines mainly rely on Bcl-xL and Mcl-1, with certain cell lines displaying increased involvement of Bfl-1. Importantly, the vemurafenib resistant SK-MEL-239 melanoma cell line showed increased functional Mcl-1 and Bfl-1 dependence compared to the parental line. Overall, this work contributes to the drug discovery efforts of Bcl-2 family inhibitors and provides novel dual Mcl-1/Bfl-1 selective inhibitors. Further optimization of these dual inhibitors may provide valuable therapeutics to help combat acquired resistance in melanoma, where Mcl-1 and Bfl-1 play prominent functional roles, as determined by BH3 profiling. Citation Format: Karson J. Kump, Lei Miao, Ahmed A. Mady, Nurul H. Ansari, Uttar K. Shrestha, Yuting Yang, Mohan Pal, Chenzhong Liao, Andrej Perdih, Fardokht A. Abulwerdi, Krishnapriya Chinnaswamy, Jennifer L. Meagher, Jacob M. Carlson, May Khanna, Jeanne A. Stuckey, Zaneta Nikolovska-Coleska. Discovery of small molecule Mcl-1 and Bfl-1 inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-226.

The CARMA1/BCL10/MALT1 (CBM) signaling complex mediates antigen receptor-induced activation of NF-kB in lymphocytes to support normal adaptive immunity. As the effector protein of the complex, MALT1 exhibits two activities: protease and scaffolding activities. Gain-of-function mutations in the CARMA1 moiety or its upstream regulators trigger antigen-independent assembly of oligomeric CBM complexes, leading to constitutive activation of MALT1, unregulated NF-kB activity, and development of Activated B-Cell subtype of Diffuse Large B-Cell Lymphoma (ABC-DLBCL). Existing MALT1 inhibitors block only MALT1 protease activity, causing incomplete and unbalanced inhibition of MALT1, and have the potential for inducing autoimmune side effects. Since MALT1 is recruited to the CBM complex via its interaction with BCL10, we sought to identify inhibitors of BCL10-MALT1 interaction in order to target both the protease and scaffolding activities of MALT1 to treat ABC-DLBCL. Our previous work suggested that an antibody-epitope-like interface governs the interaction between BCL10 and MALT1, so that a therapeutic opportunity exists for developing a small molecule inhibitor of the interaction to terminate inappropriate CBM activity. Using co-immunoprecipitation studies, a mammalian two-hybrid system, and surface plasmon resonance (SPR), we confirmed that BCL10 residues 107-119 and the tandem Ig-like domains of MALT1 are critical for this interaction. We then performed a structure-guided in silico screen of 3 million compounds, based on a computational model of the BCL10-MALT1 interaction interface, to identify compounds with potential for disrupting the interaction. Compound 1 from the initial screening hits showed dose-responsive inhibition of BCL10-MALT1 interaction in both SPR and ELISA-based assays. Functionally, Compound 1 inhibits both MALT1 protease and scaffolding activities in Jurkat T cells, as demonstrated by its inhibition of CD3/CD28-induced RelB and N4BP1 cleavage, and inhibition of IKK phosphorylation, respectively. Compound 1 also blocks IL-2 transcription and IL-2 secretion by PMA/ionomycin-treated Jurkat T cells, as well as constitutive CBM-dependent secretion of IL-6 and IL-10 by ABC-DLBCL cells. Accordingly, Compound 1 selectively suppresses the growth of ABC-DLBCL cell lines, but does not affect the growth of MALT1-independent GCB-DLBCL cell lines. In conclusion, we have identified an early-stage small molecule compound that inhibits the BCL10-MALT1 interaction, MALT1 protease and scaffolding activities, downstream CBM-dependent signaling, and ABC-DLBCL cell growth. Structure-guided modification of this lead compound is underway to further develop a new class of protein-protein interaction inhibitors that could provide more efficacious blockade of MALT1, while offering protection from undesirable autoimmune side effects in the treatment of this aggressive form of lymphoma. Citation Format: Heejae Kang, Dong Hu, Marcelo Murai, Ahmed Mady, Bill Chen, Zaneta Nikolovska-Coleska, Linda M. McAllister-Lucas, Peter C. Lucas. Inhibition of BCL10-MALT1 interaction to treat diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3404.

Effective practice of precision medicine in oncology relies on identifying and targeting unique characteristics of individual cancers. Coupling functional analysis with genomic screening will further enhance the selection of efficacious cancer therapies. Cancer is a heterogeneous disease that is defined by distinct capabilities, one of which is the evasion of apoptosis, controlled by the antiapoptotic protein members, Bcl-2, Bcl-xL, Mcl-1, and Bfl-1. These antiapoptotic proteins have become validated therapeutic targets, as manifested by the FDA-approved venetoclax, a selective Bcl-2 inhibitor, and several other molecules in clinical trials that target Mcl-1 and Bcl-xL. One of the translational challenges lies in predicting functional antiapoptotic dependence in a patient’s cancer and discriminating responders from nonresponders to Bcl-2 family targeted therapy. We have successfully characterized an updated array of Bcl-2 family antagonists, consisting of peptides and selective small-molecule BH3-mimetics via in vitro binding assays and functional analysis using model cell lymphoma cell lines. With this selective set of chemical tools, we employed the BH3 profiling assay across a wide spectrum of hematologic and solid tumor cell lines in order to functionally dissect survival dependence. To validate the results of BH3 profiling, we performed treatments with BH3-mimetics and analyzed their ability to induce apoptosis, which further demonstrated how cancers can be differentiated and targeted based on antiapoptotic dependence. The obtained data suggest that hematologic cancer cell lines mainly rely on Mcl-1 and Bcl-2 for survival and commonly undergo apoptosis in response to single-agent inhibitors of these proteins. Solid tumor cell lines are more dependent upon Bcl-xL and Mcl-1 and certain cell lines display increased involvement of Bfl-1. These cell lines undergo apoptosis when exposed to a combination of Bcl-xL/Mcl-1 inhibitors. Single agent-responsive solid tumor cell lines are less common but can be identified by BH3 profiling. This approach was successfully applied to primary patient samples to validate clinical utility. Further applications of dynamic BH3 profiling were explored to demonstrate how various FDA-approved therapies can alter antiapoptotic dependencies and aid in the design of rational combination therapies with BH3-mimetics. This work lays a translational foundation in the field of precision medicine by incorporating Bcl-2 protein family targeting into potential personalized cancer treatments. Citation Format: Karson J. Kump, Matthew Lieberman, Rita A. Avelar, Charles Foucar, Malathi Kandarpa, Antonio Di Cristofano, Russell J. Ryan, Sami N. Malek, Ryan A. Wilcox, Dale L. Bixby, Tycel J. Phillips, Moshe Talpaz, Analisa DiFeo, Zaneta Nikolovska-Coleska. Precision medicine strategy to elucidate and target Bcl-2 prosurvival proteins in a wide spectrum of cancers [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 10.

Disruptor of Telomeric Silencing 1-Like (DOT1L), the sole histone H3 lysine 79 (H3K79) methyltransferase, is required for leukemogenic transformation in a subset of leukemias bearing chromosomal translocations of the Mixed Lineage Leukemia (MLL) gene, as well as other cancers. Thus, DOT1L is an attractive therapeutic target and discovery of small molecule inhibitors remain of high interest. Herein, we are presenting screening results for a unique focused library of 1,200 nucleoside analogs originally produced under the aegis of the NIH Pilot Scale Library Program. The complete nucleoside set was screened virtually against DOT1L, resulting in 210 putative hits. In vitro screening of the virtual hits resulted in validation of 11 compounds as DOT1L inhibitors clustered into two distinct chemical classes, adenosine-based inhibitors and a new chemotype that lacks adenosine. Based on the developed DOT1L ligand binding model, a structure-based design strategy was applied and a second-generation non-nucleoside DOT1L inhibitors was developed. Newly synthesized compound 25 was the most potent DOT1L inhibitor in the new series with an IC50 of 1.0 μM, showing 40-fold improvement in comparison with hit 9 and exhibiting reasonable on target effects in a DOT1L dependent murine cell line. These compounds represent novel chemical probes with unique non-nucleoside scaffold that bind and compete with the SAM binding site of DOT1L, thus providing foundation for further medicinal chemistry efforts and developing more potent compounds.

[Image: see text] MLL-fusion proteins, AF9 and ENL, play an essential role in the recruitment of DOT1L and the H3K79 hypermethylation of MLL target genes, which is pivotal for leukemogenesis. Blocking these interactions may represent a novel therapeutic approach for MLL-rearranged leukemia. Based on the 7 mer DOT1L peptide, a class of peptidomimetics was designed. Compound 21 with modified middle residues, achieved significantly improved binding affinities to AF9 and ENL, with K(D) values of 15 nM and 57 nM, respectively. Importantly, 21 recognizes and binds to the cellular AF9 protein and effectively inhibits the AF9-DOT1L interactions in cells. Modifications of the N- and C-termini of 21 resulted in 28 with 2-fold improved binding affinity to AF9 and much decreased peptidic characteristics. Our study provides a proof-of-concept for development of nonpeptidic compounds to inhibit DOT1L activity by targeting its recruitment and the interactions between DOT1L and MLL-oncofusion proteins AF9 and ENL.

Protein-protein interactions (PPIs) represent important and promising therapeutic targets that are associated with the regulation of various molecular pathways, particularly in cancer. Although they were once considered “undruggable,” the recent advances in screening strategies, structure-based design, and elucidating the nature of hot spots on PPI interfaces, have led to the discovery and development of successful small-molecule inhibitors. In this report, we are describing an integrated high-throughput and computational screening approach to enable the discovery of small-molecule PPI inhibitors of the anti-apoptotic protein, Mcl-1. Applying this strategy, followed by biochemical, biophysical, and biological characterization, nineteen new chemical scaffolds were discovered and validated as Mcl-1 inhibitors. A novel series of Mcl-1 inhibitors was designed and synthesized based on the identified difuryl-triazine core scaffold and structure-activity studies were undertaken to improve the binding affinity to Mcl-1. Compounds with improved in vitro binding potency demonstrated on-target activity in cell-based studies. The obtained results demonstrate that structure-based analysis complements the experimental high-throughput screening in identifying novel PPI inhibitor scaffolds and guides follow-up medicinal chemistry efforts. Furthermore, our work provides an example that can be applied to the analysis of available screening data against numerous targets in the PubChem BioAssay Database, leading to the identification of promising lead compounds, fuelling drug discovery pipelines.