DLBCL Lines Research Articles

Targeting aggressive B-cell lymphomas through pharmacological activation of the mitochondrial protease OMA1.

DLBCL are aggressive, rapidly proliferating tumors that critically depend on the ATF4-mediated integrated stress response (ISR) to adapt to stress caused by uncontrolled growth, such as hypoxia, amino acid deprivation and accumulation of misfolded proteins. Here we show that ISR hyperactivation is a targetable liability in DLBCL. We describe a novel class of compounds represented by BTM-3528 and BTM-3566, that activate the ISR through the mitochondrial protease OMA1. Treatment of tumor cells with compound leads to OMA1-dependent cleavage of DELE1 and OPA1, mitochondrial fragmentation, activation of the eIF2α-kinase HRI, cell growth arrest and apoptosis. Activation of OMA1 by BTM-3528 and BTM-3566 is mechanistically distinct from inhibitors of mitochondrial electron transport, as the compounds induce OMA1 activity in the absence of acute changes in respiration. We further identify the mitochondrial protein FAM210B as a negative regulator of BTM-3528 and BTM-3566 activity. Overexpression of FAM210B prevents both OMA1 activation and apoptosis. Notably, FAM210B expression is nearly absent in healthy germinal-center B-lymphocytes and in derived B-cell malignancies, revealing a fundamental molecular vulnerability which is targeted by BTM compounds. Both compounds induce rapid apoptosis across diverse DLBCL lines derived from activated B-cell, germinal center B-cell, and MYC-rearranged lymphomas. Once-daily oral dosing of BTM-3566 resulted in complete regression of xenografted human DLBCL SU-DHL-10 cells and complete regression in 6 of 9 DLBCL patient-derived xenografts. BTM-3566 represents a first- of-its kind approach of selectively hyperactivating the mitochondrial ISR for treating DLBCL.

Abstract 3427: ICP-490 is a highly potent and selective IKZF1/3 degrader with robust anti-tumor activities against multiple myeloma and non-Hodgkin’s lymphoma

Abstract Background: Cereblon (CRBN)-targeting immunomodulatory drugs (IMiDs), i.e., thalidomide and lenalidomide, play pivotal roles in treating multiple myeloma (MM) patients. This class of drugs induce rapid ubiquitination and degradation of two lymphoid transcription factors (IKZF1 and IKZF3) by recruiting them to CRBN-CRL4 E3 ubiquitin ligase, which result in pleiotropic anti-tumor effects including autonomous tumor cell death and immune activation. However, prolonged IMiD treatment eventually becomes resistant and leads to tumor relapse in MM patients, mostly due to down-regulation of CRBN. All approved IMiDs also exert adverse effects such as teratogenicity, attributed to CRBN-mediated degradation of neo-substrates SALL4, PLZF1 and ΔNp63/TAp63. Here we report the development of a novel selective IKZF1/3 degrader ICP-490, which can robustly inhibit MM and NHL tumor growth, and overcome lenalidomide resistance. Results: ICP-490 induces potent, deep, and selective degradation of IKZF1 and IKZF3 with sub-nanomolar IC50, but no significant degradation of IKZF2, IKZF4, GSPT1, SALL4 and PLZF1. Cell viability assays reveal robust in vitro efficacies of ICP-490 against various MM and NHL(DLBCL) cell lines with nanomolar IC50. It also exhibits potent anti-proliferative activity in lenalidomide-resistant cell lines. In contrast to its tumor killing effect, ICP-490 shows no cytotoxicity against normal human cells, such as HEK293 and PBMC cells. ICP-490 induces IKZF1/3 degradation in distinct human lymphocyte lineages, leads to immune cell activation with elevated IL-2 and IFN-γ production and down-regulation of TNF-α in PBMC. The immune modulation activity of ICP-490 has also been illustrated in a combinatory treatment with daratumumab, where low dose of ICP-490 leads to robust induction of IL-2 and granzyme B, and much improved efficacy of daratumumab in MM cell line NCI-H929. In vivo efficacy studies have further confirmed the effectiveness of ICP-490 against MM and DLBCL xenografts, including MM1.S, MM1.R, NCI-H929 multiple myeloma lines, and TMD-8, WSU-DLCL2 DLBCL lines. ICP-490 also eradicates lenalidomide-resistant NCI-H929 xenografts. ICP-490 has overall favorable pharmacokinetic parameters with high oral bioavailability. Nonclinical safety evaluations of ICP-490 exhibit acceptable drug tolerability in SD rats and cynomolgus monkeys. Conclusions: ICP-490 is a highly potent, orally bioavailable, next-generation CRBN-targeting IMiD with selective IKZF1/3 degradation. In various MM and NHL tumor models, it demonstrates superior tumor killing activities than currently approved IMiDs, including overcoming lenalidomide resistance. ICP-490 is now in phase I clinical trial. Citation Format: Ruixia Liang, Yucheng Pang, Yingrui Han, Haipeng Xu, Zuopeng Wang, Richard Liu, Charles Ying Wang, Jason Bin Zhang, Xiangyang Chen, Davy Xuesong Ouyang. ICP-490 is a highly potent and selective IKZF1/3 degrader with robust anti-tumor activities against multiple myeloma and non-Hodgkin’s lymphoma [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 3427.

Enhancing α-CD19 CAR T Antitumor Efficacy Using the BH3 Mimetic, Venetoclax

Introduction Clinical outcomes of anti-CD19 chimeric antigen receptor T cell (CAR T) therapy for the treatment of relapsed/refractory Diffuse Large B Cell lymphoma (r/r DLBCL) must be improved, as patients undergoing CAR T therapy can expect a cure rate of 41% and median survival of just 6.3 months if unresponsive. Poor clinical responses to CAR T therapy and a small window of survival highlights the critical need for improvement of CAR T therapy in r/r DLBCL. Our lab previously revealed synergy between the Bcl-2 inhibitor, Venetoclax, and α-CD19 CAR Ts. Though the role of Bcl-2 inhibition within B cell lymphomas is well characterized, further investigation is needed to understand its impact within effector cell populations. Our lab previously established a role for Bcl-2 inhibition in promoting the differentiation of bulk CD8+ CAR T into CD8+ CAR TStem Cell Memory (TSCM) subsets, decreasing the frequency of CD4+ CAR TRegulatory subsets, and increasing cytotoxicity of CAR T when co-cultured with rituximab/chemoresistant malignant B cells, Raji4RH. Here, our lab further elucidates the molecular mechanisms driving these improvements in CAR T quality as a consequence of Bcl-2 inhibition. MethodsTransduction was performed on healthy donor T cells with lentiviral vectors encoding for 2nd generation α-CD19 CAR constructs containing CD28 (28ζ) or 4-1BB (BBζ) costimulatory domain, 24 hours after activation with αCD3/CD28 TransACT (Miltenyi). Cytotoxicity assays were performed by labeling target cells with a lipophilic dye (Sigma-Aldrich), then incubated with GFP+ CAR T at various effector:target ratios and Venetoclax concentrations for 24 hours in 96 well U-bottom plates. Sample wells were stained with viability dye and acquired on BD LSR II cytometer. Mitochondrial membrane potential (ΨM) was assessed using a cell-permeant fluorescent probe, TMRM (Thermofisher), which was added to immunophenotype panels. CAR T immunophenotype was identified using markers for viability, CD3, CD4, CD8, CD45RA, CD45RO, CD62L, CCR7, CD25, CD95, and TMRM. Frequencies of T cell memory subsets were calculated alongside changes in ΨM. B-cell marker expression was assessed for several ABC and GCB DLBCL lines. Antibodies against CD10, CD20, CD22, CD138, and CD19 were used to establish B-lineage epitope density within our target populations. Proliferation tracking was performed using cell-permeant dye, CellTrace Blue (Thermofisher). Bulk untransduced, CD28, or 4-1BB CAR T were stained with tracking dye, activated for 24 hours by co-culture with CD19+ cells, then isolated using CD19+ Microbead positive selection (Miltenyi Biotec). Then, the untransduced control and CAR T fractions were cultured in Venetoclax for up to 96 hours. Samples were then collected and stained with naïve and memory T cell markers and a viability dye. Results Untransduced control, 28ζ, and BBζ CAR T were exposed to Venetoclax during co-culture with DLBCL lines expressing varying densities of CD19 epitope. A significant concentration-dependent decrease in ΨM was observed for each CAR construct. CAR viability did not decrease more than 5% among each concentration and cell line tested. As Venetoclax concentration increases, larger frequencies of CD8+ TNaive and TSCM are observed. Therefore, we conclude an inverse relationship between ΨM and differentiation into CD8+ TSCM cells. CD19+ epitope density does not significantly impact CAR memory phenotype, ΨM, or cytolytic capacity. To uncover the progenitor cell type populating Venetoclax-exposed CD8+ CAR TSCM cells, we tracked dye loss between subsets TNaive, TSCM, and TTotal. After 96 hours of culture, it was apparent that the TSCM cohort was not significantly proliferating, however the TNaive subset experienced the most dye loss, and accounted for observed dye loss within the TTotal cohort. We conclude that Venetoclax encourages differentiation from TNaive cell to TSCM cell. Conclusion We reveal intrinsic changes within the CAR T as a result of Bcl-2 inhibition; and propose that these changes are due to manipulation of mitochondria, allowing for differentiation into a more quiescent, stem-like cell type. Low ΨM has been linked to memory T cell phenotype and increased anti-tumor function in vivo. We seek to uncover the metabolic mechanisms which regulate the observed changes within CAR Ts as a result of Bcl-2 inhibition. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract A07: BTM 3566, a novel activator of the mitochondrial stress response promotes robust therapeutic responses in vitro and in vivo in diffuse large B-cell lymphoma

Abstract Relapsed/refractory diffuse large B-cell lymphomas (r/r-DLBCL) are a therapeutic challenge as they are highly heterogeneous both clinically and molecularly, which imposes a pressing need to develop novel therapies to improve outcomes in patients independently of the molecular subtype. We describe here BTM-3566, a first-in-class, orally active compound with activity against DLBCL. BTM-3566 induces apoptosis and complete cell killing in DLBCL lines a with an IC50 of ~200 – 500 nM. Responsive DLBCL cell lines include ABC, GCB, and double-hit and triple-hit lymphoma type. Pharmacokinetic studies in mice showed suitability for once daily dosing, with > 50% oral bioavailability and ~6 hours of serum half-life. In xenograft models using the double-hit DLBCL line SU-DHL-10, BTM-3566 treatment resulted in complete response in all tumor-bearing animals and durable remission in 50% of animals with no tumor growth occurring for 2 weeks after dose cessation. Expansion studies into human DLBCL PDX models harboring a range of high-risk genomic alterations demonstrated response in 100% of the lines with complete response in 6 of 9 PDX models tested. Dose scheduling studies indicate that 7 days of continuous dosing is sufficient to induce tumor regression and maintenance of a complete response. The therapeutic activity of BTM-3566 is related to a novel effect on mitochondrial proteostasis and a robust induction of the ATF4 ISR. Of the four eIF2α -kinases in the human genome we determined that HRI was uniquely required for BTM-3566 activity in DLBCL. HRI is activated by mitochondrial-related stress resulting in activation of the mitochondrial protease OMA1. Molecular analysis revealed that BTM-3566 activates OMA1, leading to induction of the ATF4 ISR pathway and apoptosis in DLBCL cell lines. Deletion of OMA1 reduces the ability of BTM-3566’s to induce apoptosis in DBLCL. Substrates of OMA1 include the dynamin OPA1 and DELE1, a protein recently shown to signal mitochondrial dysfunction through activation of HRI kinase and ATF4. Transfection of BJAB cells with a cleavage resistant OPA1 mutant has no effect on BTM-3566 induced apoptosis. In contrast, DELE1 KO suppresses BTM-3566 mediated apoptosis. BTM-3566 activates OMA1 without acting as a classical mitochondrial toxin. Instead, BTM-3566 induces OMA1 activity through a novel mechanism regulated by the mitochondrial protein FAM210B. FAM210B expression is negatively correlated with response to BTM-3566, and overexpression of FAM210B blocks OMA1 activation and causes complete resistance to BTM-3566 induced apoptosis. Taken together, these data support a novel antitumor mechanism in DLBCL, where BTM3566 induces mitochondrial stress, activating the OMA1-DELE1-HRI-eIF2a-ATF4 pathway leading to apoptosis and tumor regression. An Investigational New Drug application for BTM3566 in B-cell malignancies will be submitted in Q2 2022 with initiation of first in human clinical trials planned for fall 2022. Citation Format: Adrian Schwarzer, Matheus Oliveira, Marc-Jens Kleppa, Andy Anantha, Alan Cooper, Mark Hannink, Todd Hembrough, Jedd Levine, Michael Luther, Michael Stocum, Linsey Stiles, Marc Liesa-Roig, Matthew Kostura. BTM 3566, a novel activator of the mitochondrial stress response promotes robust therapeutic responses in vitro and in vivo in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A07.

P1291: BTM-3566, A NOVEL ACTIVATOR OF THE MITOCHONDRIAL STRESS RESPONSE ERADICATES DIFFUSE LARGE B-CELL LYMPHOMA IN MICE

Background: Relapsed/refractory diffuse large B-cell lymphomas (r/r-DLBCL) are a therapeutic challenge, especially in patients not suitable for high dose chemotherapy, stem cell transplantation or after CAR-T-cell therapy. Aims: We describe and characterize BTM-3566, a first-in-class compound that activates the OMA1-dependent mitochondrial stress pathway. BTM-3566 is active against a variety of B-cell malignancies but has the greatest effect in DLBCL, inducing robust therapeutic responses in vitro and in vivo. Methods: Chemical biology, animal models, functional genetics Results: BTM-3566 is a small molecule based on a pyrazolothiazol-backbone. BTM-3566 induces apoptosis and complete cell killing in DLBCL lines with an IC50 of ~300 nM, including ABC, GCB, double-hit and triple-hit lymphoma lines. BTM-3566 has > 50% of oral bioavailability and 6 hours of serum half-life. 14-day dosing in mice and dogs demonstrated excellent tolerability at therapeutic doses. In a dose-finding study using the DLBCL line SUDHL-10, once daily oral treatment with 20 mg/kg BTM-3566 for 21 days resulted in complete regression in all tumor-bearing animals. Importantly, no subsequent tumor growth was observed for 2 weeks after cessation of therapy. Expansion studies into human DLBCL PDX models harboring a range of high-risk genomic alterations, demonstrated response in 100% of the lines with complete tumor regression in 6 of 8 PDX models tested (Table 1). Transcriptome analyses revealed that BTM-3566 activates the ATF4-integrated stress response (ISR), indicated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and upregulation of the stress transcription factor ATF4. Of the four eIF2a-kinases in the human genome we determined that HRI (EIF2AK1) was uniquely required for BTM-3566 eIF2a phosphorylation, activation of the ATF4-ISR and induction of apoptosis. HRI is activated by mitochondrial-related stress, including heme depletion, ROS generation or blockage of mitochondrial ATP synthesis which all result in the activation of the mitochondrial protease OMA1. We found that BTM-3566 activates OMA1 in a manner unrelated to changes in mitochondrial ATP synthesis, reactive oxygen species generation or electron transport chain inhibition. OMA1 activation was required for the therapeutic effect and CRISPR-Cas9 depletion of OMA1 eliminated BTM-3566’s apoptotic activity. Substrates of OMA1 include the mitochondrial GTPase OPA1 and DELE1, a mitochondrial protein recently shown to act as a sensor of mitochondrial dysfunction and to signal through HRI kinase and ATF4. We show that downstream of OMA1 the apoptotic stress signal is relayed to HRI by DELE1, whereas OMA1-dependent OPA1 cleavage and mitochondrial fragmentation were dispensable for the therapeutic effect. Gene expression-based profiling of BTM-3566 sensitivity in over 400 cancer cell lines showed that FAM210B, a poorly characterized mitochondrial membrane protein, negatively correlated with response to BTM-3566. Overexpression of FAM210B prevented OMA1 activation and causes complete resistance to BTM-3566-induced apoptosis and cell cycle arrest. Thus, FAM210B serves as a strong predictor of BTM-3566 sensitivity, as well as revealing a novel mechanism of regulation of OMA1 activation. Image:Summary/Conclusion: We describe a novel antitumor mechanism in DLBCL, where BTM-3566 induces mitochondrial stress, activating the OMA1-DELE1-HRI-eIF2a-ATF4 pathway leading to apoptosis and tumor regression. An IND application in DLBCL has been completed with initiation of first in human clinical trials planned in 2022.

BCL10 Gain-of-Function Mutations Aberrantly Induce Canonical and Non-Canonical NF-Kb Activation and Resistance to Ibrutinib in ABC-DLBCL

ABC-DLBCLs are among the most aggressive DLBCLs. Genome sequencing studies identified BCL10 gain-of-function mutations in DLBCL mostly within the ABC-DLBCLs. ABC-DLBCLs were recently split into further subtypes according to their genomic characteristics, among which BCL10 somatic mutations were most common in BN2/Cluster 1 cases. These are likely transformed marginal zone lymphomas and were recently suggested to be sensitive to ibrutinib treatment. Pooling publicly available data we noted that most BCL10 mutations are missense or nonsense SNVs affecting its CARD domain, MALT1 binding domain, and the C-terminal S/T rich domain. We generated a large panel of mutant BCL10 constructs including the top hotspot missense mutation R58Q (CARD domain) and nonsense truncation mutations such as E140X. We found that almost all mutants induced aberrantly strong induction of NF-κB activity in lymphoma cells as compared to WT BCL10, indicating that they induce gain of function. BCL10 forms a high order complex with CARD11 and MALT1 (CBM signalosome) downstream of BCR signaling. Normally, CARD11 activation induces polymerization of BCL10 which induces MALT1 activity and downstream NF-κB signaling. To investigate the impact of BCL10 mutants on CBM complex formation we performed fluorescence polarization and filamentation formation assays with purified WT and mutant BCL10 species. Both BCL10R58Q and BCL10E140X manifested faster and even spontaneous polarization compared to BCL10WT. BCL10R58Q formed thicker and more heavily bundled filaments (~20 nm) that provide greater surface area to dock signaling proteins, whereas filaments formed by BCL10E140X had the normal ~10 nm structure. Even though the BCL10E140X deletes the canonical MALT1 binding site, the mutant filament still featured robust MALT1 recruitment. Cryo-EM studies revealed that BCL10R58Q mutant gains new interactions within the filament structure that could explain the observed stabilization and bundling effects. Notably, cryo-EM structure of the BCL10E140X mutant in complex with MALT1 showed that it retains its interaction with MALT1 in the filament form despite its predicted lack of interaction with MALT1 in the monomeric form due to the C-terminal deletion. To gain further functional insight we performed mass spectrometry to identify proteins interacting with WT, BCL10R58Q and BCL10E140X. BCL10R58Q featured gain of many novel protein interactors including NF-κB2 and TAB1 etc. consistent with bundled filament formation enabling more signaling protein recruitment. However, the BCL10E140X interactome was quite different and most notably featured loss of binding to negative regulators of non-canonical NF-κB. NF-κB2 (p100/p52) level was indeed elevated in the presence of this mutant. In addition, both BCL10 mutant classes showed aberrant activation of canonical and non-canonical NF-κB activation (IkBa/p65 and p52) through distinct mechanisms. As a functional readout of BCL10 function, we generated a MALT1 GloSensor reporter DLBCL lines to detect MALT1 protease activity. Indeed, both classes of mutations showed potent induction of MALT1 protease activity (GloSensor), enhanced cleavage of canonical MALT1 target proteins (Western Blot), expression of canonical NF-κB target genes (QPCR) such as IL6 and IL10. In striking contrast to BCL10WT, and consistent with our structural data showing spontaneous polymerization of BCL10 mutants, we found that CARD11 knockdown did not impair MALT1 activation, NF-κB signaling, or cell growth in ABC-DLBCL lines expressing both BCL10 mutants. This CARD11 independence was concerning, since it suggests that BCL10 mutant lymphomas might be resistant to drugs targeting upstream components of the BCR signaling pathway such as ibrutinib. Indeed, expression of BCL10R58Q and BCL10E140X (but not BCL10WT) in various ABC-DLBCL cell lines abrogated the ability of ibrutinib to inhibit MALT1 (GloSensor), NF-κB activity (Reporter), cell growth (Growth inhibition) as well as proliferation. Collectively, we find that BCL10 mutations induce aberrant canonical and non-canonical NF-κB activity through novel and structurally distinct biochemical mechanisms that are at least partially dependent on MALT1. BCL10 mutation should be considered as a biomarker for ibrutinib resistance in ABC-DLBCL, so that alternative targeted therapies can be prioritized for these patients. Disclosures Fontan: Johnson & Johnson: Current Employment. Melnick:Epizyme: Consultancy; Jubilant: Consultancy; Constellation: Consultancy; Janssen: Research Funding; Daiichi Sankyo: Research Funding.

Abstract LB-272: KYM-001, a first-in-class oral IRAK4 protein degrader, induces tumor regression in xenograft models of MYD88-mutant ABC DLBCL alone and in combination with BTK inhibition

Abstract Purpose: This work assessed the antitumor activity of selective small molecule IRAK4 degraders in human ABC DLBCL cell lines in vitro and in tumor xenograft models in vivo, alone and in combination with BTK inhibition. Introduction: ABC DLBCL comprises approximately 45% of DLBCL and has a worse outcome with R-CHOP chemotherapy compared to GCB DLBCL. Activating mutations in MYD88 occur in 30-40% of ABC DLBCL; L265P, the most prevalent MYD88 mutation, causes constitutive assembly and activation of the Myddosome. IRAK4 kinase and scaffolding functions are essential for full signaling through the Myddosome to NFκB and MAPK pathways. Kymera Therapeutics is using a chemical knockdown strategy to develop heterobifunctional small molecule IRAK4 degraders, exemplified by KYM-001, for the treatment of MYD88-driven lymphomas. Methods: IRAK4 in human PBMC, ABC DLBCL cell lines and xenografts was quantified by immunoassays or targeted MS/MS. Myddosome signaling was monitored by mRNA and phosphoprotein endpoints. Cell viability and cell cycle were monitored by flow cytometry. Tumor xenograft studies were conducted by implanting human ABC DLBCL lines into immunocompromised mouse strains and assessing tumor volume. Key data: KYM-001 led to potent E3 ligase-dependent degradation of IRAK4. Notably, KYM-001 more effectively inhibited TLR-activated Myddosome signaling compared to IRAK4 kinase inhibitors in human PBMC. Degradation was highly selective for IRAK4 vs >10,000 other detected proteins in the MYD88 L265P mutant ABC DLBCL line OCI-LY10. IRAK4 degradation by KYM-001 resulted in cell cycle inhibition and apoptosis within 48-72 h in ABC DLBCL, with preferential activity in MYD88-mutant vs MYD88-WT cell lines. Oral dosing of KYM-001 showed dose-dependent antitumor activity in several mouse xenograft models of human MYD88-mutant ABC DLBCL at tolerated doses and schedules. In the OCI-LY10 model, tumor regression was associated with >80% degradation of IRAK4, establishing the pharmacodynamic effect required for maximal efficacy. Since alterations in BCR signaling and MYD88 frequently co-occur in B-cell malignancies, we investigated the potential for combined activity of IRAK4 degradation and BTK inhibition. In the OCI-LY10 xenograft model, which has activating mutations in both CD79B and MYD88, BTK inhibition with ibrutinib had an additive effect on KYM-001 antitumor activity. Conclusions: KYM-001 is a first-in-class, potent, selective and orally active IRAK4 degrader that causes tumor regression in ABC-DLBCL models. Degradation of IRAK4 removes both the kinase and scaffolding functions of IRAK4, and may be superior to kinase inhibition alone. These data support IRAK4 degraders as a promising new therapeutic opportunity for MYD88-driven lymphoma, both alone and in combination with other targeted approaches such as BTK inhibition. Citation Format: Joseph F. Kelleher, Veronica Campbell, Jesse Chen, Jared Gollob, Nan Ji, Hari Kamadurai, Christine Klaus, Henry Li, Christine Loh, Alice McDonald, Haojing Rong, Scott Rusin, Kirti Sharma, Dominico Vigil, Duncan Walker, Matt Weiss, Karen Yuan, Yi Zhang, Laurent Audoly, Nello Mainolfi. KYM-001, a first-in-class oral IRAK4 protein degrader, induces tumor regression in xenograft models of MYD88-mutant ABC DLBCL alone and in combination with BTK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-272.

Abstract 346: Potential role for R191, potent and selective IRAK4 kinase inhibitor, in treatment of hematologic malignancies

Abstract Recent advances in genome sequencing and tumor proteome and transcriptome analysis uncovered a key role for MyD88-dependent Toll-Like Receptor (TLR) and Interleukin-1 Receptor (IL-1R)-mediated signaling pathways in multiple hematologic malignancies. A third of activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) and nearly 100% of Waldenstrom macroglobulinemia (WD) patients carry an activating Myd88 L265P mutation. Overexpression of multiple TLR pathway components, associated with deregulation of innate immune system and subsequent induction of proinflammatory bone marrow environment, are prominent features of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). IRAK4 kinase is a crucial enzyme in all MyD88-dependent signaling pathways, potentially making it an ideal target for disease modification with small molecule inhibitors. Through cell-based screening, we identified a potent small molecule IRAK1/4 kinase inhibitor, R191. R191 blocks TLR- and IL-1R-induced cytokine production in primary cells with potencies below 50nM while sparing unrelated pathways with at least 20-fold window. R191 is extremely potent in vitro against IRAK4 kinase (3 nM), yet exhibits good selectivity against a broad panel of kinases. In vivo, it decreases serum IL-6 in an acute mouse model of IL-1β-induced cytokine release and blocks joint inflammation in the collagen-induce arthritis model. R191 exhibited strong synergy with Bcl2 and BCR pathway inhibitors against a number of DLBCL lines in vitro. In multiple AML lines, R191 potently inhibited expression of PD-L1, potentially promoting recognition of AML blasts by the immune system. Based on the potential critical role of IRAK kinases in MDS, AML and Myd88 L265P lymphomas, R191 could provide a novel therapeutic modality in the management of multiple inflammation-driven hematologic malignancies. Citation Format: Vadim V. Markovtsov, Chrystelle Lamagna, Meagan Chan, Sothy Yi, Chi Young, Roy Frances, Stacey Siu, Sylvia Braselmann, Hui Li, Rajinder Singh, Gary Park, Esteban Masuda, Vanessa Taylor, Donald G. Payan. Potential role for R191, potent and selective IRAK4 kinase inhibitor, in treatment of hematologic malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 346.

The PI3 Kinase (PI3K) Inhibitor, Buparlisib/BKM120, in B-Cell, T-Cell and Hodgkin Lymphoma Cell Lines: Cell Death Studies and Examination of Genomic Pathway Analyses Via a Systems Biology Approach

Introduction: The PI3K pathway plays a significant role in cell cycle, apoptosis, and DNA repair. It is commonly dysregulated in cancers making it a potential therapeutic target. BKM120 is a novel oral pan-class I PI3K inhibitor with antitumor activity and efficacy reported in solid tumors. It is in phase I/II clinical trials for treatment of relapsed-refractory NHL. There are additional data desired towards molecular mechanism of action and biological pathways of resistance for BKM120-treated lymphoma.Methods: TCL cell lines (Jurkat, Hut78, HH), HL lines (L428, L540) and DLBCL lines (SUDHL4, SUDHL6, SUDHL10, OCILY3, OCILY19) were treated with increasing concentrations of BKM120 (0.16-10uM) in 96 well plate and cell viability assessed by MTT assay. For gene expression profiling (GEP), SUDHL6, OCILY3, Jurkat, Hut78 and L540 cells were treated at IC50and analyzed on Illumina human HT12 gene chip. Gene Set Enrichment Analysis (GSEA) and biological network analysis were done using Ingenuity Pathway Analysis and Cytoscape. Apoptosis was evaluated by Annexin V and propidium iodine (AV/PI) staining and flow cytometry. For cell cycle analysis, Jurkat cells were exposed to BrdU and stained with anti-BrdU FITC and 7-AAD and analyzed by flow cytometry.Results: Cell viability showed dose-dependent growth inhibition in all lines. IC50 was between 0.316μM - 3.72μM at 72 hours (hrs). GEP analyses showed significant gene changes following BKM120 treatment with a 1.2 fold-change for OCILY3 (1886 genes), SUDHL6 (1884 genes), Hut78 (1474 genes), L540 (859 genes) and Jurkat (212 genes) (Fig 1). Differential gene expression with BKM120-treated cells showed significant overlap among DLBCL cell lines, with 991 genes of 1886 (OCILY3) and 1884 (SUDHL6). The differentially expressed genes in L540 HL cells treated with BKM120 showed better overlap with DLBCL lines than with TCL lines, with 366 and 315 of 859 L540 genes overlapping with OCILY3 and SUDHL6 lines, respectively. This may reflect the B cell of origin related response to BKM120 treatment. TCL lines had different profiles, with 52 overlapping genes of 212 for Jurkat and 1474 for Hut78. Jurkat is known to harbor a PTEN mutation with constitutively active PI3K, which may explain the differences in the GEP observed with BKM120 treatment in Jurkat and Hut78 TCL. GSEA and biological network analysis of BKM120 treated lymphoma cells showed conserved inhibitory effects on cell cycle, DNA replication and metabolic process across all lymphoma cells. However, we observed that immune signaling processes were oppositely regulated in B and T cell lymphomas. AV/PI-staining and flow cytometry revealed dose-dependent increase of apoptotic cells in all lymphoma cell lines. Analysis based on BrdU incorporation revealed induction of G2/M arrest in Jurkat cells treated with BKM120. Western blots showed decreased phosphorylation of PI3K and mTOR substrates including 4-EBP and phospho-p70S6K with BKM120 treatment. Although phosphorylation of MEK/ERK were downregulated at lower doses of BKM120, at higher doses, increased MEK/ERK phosphorylation were seen. Analysis of cell cycle regulatory proteins Cyclin, CDK and phospho-histone H3 with BKM120 treatment resulted in expression changes consistent with G2/M arrest and also resulted in increased apoptosis related PARP cleavage in all lymphoma cell lines.Conclusions: BKM120 treatment elicited biologically distinct GEP with immune functions oppositely regulated in B and T cell lymphomas, however impairment of cell cycle, DNA replication and metabolic function were the core responses/function to PI3K inhibition. Furthermore, based on GSEA, it was predicted that cell cycle arrest and MAPK activation as likely mechanisms of resistance to BKM120 treatment in these lymphoma cell lines. Additional studies are planned to examine rational combinations of BKM120 together with other targeted small molecules (e.g., Chk1-inhibitor, CDK4/6 inhibitor and MEK inhibitor). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Synergistic Combinations of Histone Deacetylase Inhibitors and Decitabine Induce a Unique Gene Expression and Epigenetic Profile In Models of Diffuse Large B-Cell Lymphoma

AbstractAbstract 435Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoid malignancy, representing approximately 30–40% of all lymphomas. While significant progress has been made in treating this disease over the past decade, it is still regarded as a heterogeneous disease which, after being classified as relapsed or refractory, is fatal in about one-third of patients. Histone deacetylase inhibitors (HDACI) are presently approved for the treatment of relapsed or refractory cutaneous T- cell lymphomas (CTCL), and have marked activity in peripheral T-cell lymphomas (PTCL), though their effectiveness in DLBCL is less established. DNA methyltransferases (DNMTs) are known to recruit and cooperate with histone deacetylases to induce gene silencing. Combinations of drugs affecting these pathways have emerged as active and important, mostly in myeloid leukemias. We hypothesized that the combination of HDACI and DNMT inhibitors (DNMTI) in DLBCL may be active only in combination and not as single agents. We examined the interaction between a broad range of HDACI including vorinostat, depsipeptide, panobinostat and DNMTI using in vivo and in vitro models of DLBCLs, clearly confirming that these agents are in fact synergistic with decitabine. Synergy was measured by relative risk ratio (RRR) and the values obtained were as low as 0.01, representing very strong synergy. This combination of drugs, specifically panobinostat and decitabine, was also shown to be strongly synergistic in a murine xenograft model of DLBCL. In addition, we analyzed the molecular basis for this synergistic effect by evaluating the global gene expression and methylation using microarrays on the cells treated with the single agents and combination in DLBCL. Three DLBCL lines (OCI-Ly1, OCI-Ly10 and Su-DHL6) were treated with decitabine alone (2.5 μ M), panobinostat alone (2.5 nM) or their combination for 48h hours. DNA and RNA from untreated and treated cells were used for genome wide methylation analysis through Illumina Humanmethyation27 platform and gene expression profiling analysis with Illumina HumanHT-12 v3 Expression arrays. 3D principal component analysis clearly clustered the samples treated with panobinostat and combination therapy together and at greater distances from untreated samples and samples treated by decitabine alone. Therefore, the contribution to the gene expression phenotype of the combination was greater from the HDACI than with DNMTI. Consistent with this observation, the top network of genes differentially expressed (p<0.05) by panobinostat involved critical transcription factors like GATA1, GATA4, SMAD and DNMT3A. Additionally, network-functional analysis of genes perturbed by the combination treatments enriched for critical pathways involved in cell death, cell development and cellular proliferation. Surprisingly, differentially expressed genes and networks identified by each of the treatment conditions and by combination therapy were unique with few overlapping genes as shown in Venn diagram in Figure 1a. Genome wide methylation analysis produced similar results with greater contribution to global methylation changes in cells treated by the combination therapy and decitabine as compared to HDACI. Again, methylation status of a distinct set of genes was altered by combination therapy as compared to the individual drugs (Figure 1b). Correlation between genome wide methylation analysis and gene expression profiling identified 16 overlapping genes in the samples treated by the combination of panobinostat and decitabine including known tumor suppressor genes like VHL, DIRAS3 and WT1. Taken together, integrative genomic analysis has provided insights into the relative contribution of independent epigenetic therapies to the combination phenotype. These findings may provide important leads in identifying unique biomarkers of response specific to the combination of panobinostat and decitabine in DLBCL. [Display omitted] Disclosure:O'Connor:Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

A Small Molecule PLK1 Inhibitor, MLN0905, Induces An Anti-Tumor Response In Human Models of Diffuse Large B-Cell Lymphoma

AbstractAbstract 3922Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas, accounting for up to 30 percent of newly diagnosed cases. Current treatment options for this disease are effective, but not always curative; therefore experimental therapies are being investigated. Diffuse large B-cell lymphoma cells typically over-express the serine/threonine mitotic kinase PLK1, which plays a key role in mitotic cell cycle progression, and has been linked to poor patient prognosis. We have discovered a potent and selective small molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL lines. We explored the pharmacokinetic, pharmacodynamic, and anti-tumor properties on MLN0905 in mouse models harboring human DLBCL disease. MLN0905 has drug-like pharmacokinetic properties and an acceptable toxicity profile making it a good clinical candidate. In human xenograft tumor tissue, MLN0905 modulates the pharmacodynamic biomarker phospho-Histone H3 (in a dose dependent fashion), enabling us to track pathway modulation in vivo. The anti-tumor activity of MLN0905 was evaluated in three human subcutaneous xenograft models OCI-LY10, OCI-LY19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant anti-tumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The anti-tumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI-LY19) setting to better mimic DLBCL in humans. In this disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with Rituximab in the disseminated OCI-LY19 model. Combining Rituximab and MLN0905 provided both a synergistic anti-tumor effect and a synergistic survival advantage. Our findings indicate for the first time that PLK1 inhibition leads to pharmacodynamic pH3 modulation and significant anti-tumor activity in multiple models of DLBCL. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anti-cancer agents in the clinic. Disclosures:Shi:Millennium: The Takeda Oncology Company: Employment. Lasky:Millennium: The Takeda Oncology Company: Employment. Shinde:Millennium: The Takeda Oncology Company: Employment. Duffey:Millennium: The Takeda Oncology Company: Employment. Stringer:Millennium: The Takeda Oncology Company: Employment. Qian:Millennium: The Takeda Oncology Company: Employment. Liao:Millennium: The Takeda Oncology Company: Employment. Liu:Millennium: The Takeda Oncology Company: Employment. Rao:Millennium: The Takeda Oncology Company: Employment. Vos:Millennium: The Takeda Oncology Company: Employment. D'Amore:Millennium: The Takeda Oncology Company: Employment. Hyer:Millennium: The Takeda Oncology Company: Employment.

Histone Deacetylase Inhibitors Synergistically Enhance the Cytotoxic Effect of Hypomethylating Agents in Models of Diffuse Large B-Cell Lymphoma.

Abstract 3720Poster Board III-656Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoid malignancy in the world representing 30-40% of all lymphomas. Gene expression profiling analyses from patients with DLBCL have revealed that DLBCL is a heterogeneous disease consisting of at least two subtypes: activated B-cell (ABC) like DLBCL and germinal center B (GCB) like DLBCL. Standard immunochemotherapy (R-CHOP) ensures cure in 55-60% of patients, while the remaining patients are destined to succumb to their disease. Thus, innovative strategies to treated patients with relapsed or refractory disease are urgently needed, especially for those with the ABC subtype who experience a markedly inferior overall survival. We have analyzed the cytotoxic effect of 6 histone deacetylase inhibitors (HDACi: panobinostat, vorinostat, depsipeptide, belinostat, MS-275 and Scriptaid) and 2 hypomethylating agents (decitabine and 5-azacytidine) in in vitro models of DLBCL (3 ABC DLBCL lines – OCI-Ly10, Riva, HBL-1 and 3 GCB DLBCL lines – OCI Ly1, OCI-Ly7 and SU-DHL6). As single agents HDACi exhibited IC50 values in the nanomolar range with depsipeptide and panobinostat being the most potent. The IC50 values for the hypomethylating agents was typically in the low micromolar range, with decitabine being slightly more more potent than 5-azacytidine. When combined with decitabine all HDACi exhibited a synergistic effect in inducing growth inhibition, measured by luminescence cell viability assays and apoptosis flow cytometry assays. This effect was shown by the relative risk ratio (RRR) calculation where the actual value of the combination sample is divided with the expected value, where values < 1 represent synergy. In addition, the combination of panobinostat and decitabine was synergistic in their ability to activate caspase 3, analyzed by flow cytometry, and their ability to induce histone acetylation in Western blot and immunofluorescence experiments. The results from in vitro experiments were confirmed in a murine model (SCID beige) of a human lymphoma xenograft, (Ly1 DLBCL line) where the activity of the combination of panobinostat and decitabine in two treatment schedules (both combination schedules had equal doses of panobinostat: 75 mg/kg divided in 5 daily doses, and differed only in the dose of decitabine, where one group received 4.5 mg/kg divided in three daily doses, and the second 3 mg/kg in two daily doses) was compared to single drug activity. Both combination groups were superior to single drug cohorts and control in inhibiting the tumor volume growth calculated for relative and absolute volumes and area under the tumor volume curve (p < 0.05). Interestingly, the toxicity of the combination was proportional to the decitabine dose. These experiments confirm the activity of this combination in preclinical models of DLBCL, and warrant more detail clinical studies in patients with relapsed or refractory DLBCL. Disclosures:O'Connor:Abbott: Research Funding.

Clonally Related B-NHL Populations and Cell Lines Harbor Subsets with Significant Phenotypic and Functional Heterogeneity.

Abstract 1699Poster Board I-725Many malignancies such as myeloid leukemia, breast, colon, and CNS tumors appear to harbor phenotypically and functionally distinct populations of cells with self-renewal, differentiative, and tumorigenic capacity. These unique populations, influencing tumor growth and persistence, have been termed “cancer stem cells,” or “tumor-initiating cells.” No such tumor-initiating capacity has been prospectively identified in non-Hodgkin lymphoma (NHL). Identification of these novel sub-populations in B-NHL is particularly challenging given the unique proliferative capacity, differentiation potential, and population diversity of the peripheral B cell subsets from which lymphomas arise. Our studies have focused on the identification of sub-populations that meet criteria for “lymphoma stem cells” (LySC) in B-NHL samples. Two experimental sources of NHL cells were used: i) primary human DLBCL (TXN190, TXN131, C10, C12) derived at our institution from malignant effusions, and, ii) continuously cultured, widely used DLBCL lines (OCI-Ly-7, -10, -19).Using multiparameter FACS, we initially performed immunophenotypic studies to investigate a broad range of cell surface markers. Notably, we observed significant heterogeneity in the surface antigen density for CD22, CD27, CD45R/B220, and CXCR5. These data suggest that subpopulations residing within NHL populations may possess functional heterogeneity. To test this concept, we first performed colony-forming unit (CFU) assays in methycellulose medium. Analysis of 7 specimens shows a CFU frequency ranging from .0003% to 10% of the overall population, demonstrating functional heterogeneity with regard to in vitro colony-forming ability. Surprisingly, we found that generation and successful re-plating of B-NHL colonies did not require the addition of exogenous factors such as PHA-conditioned medium, cytokine, or interleukin supplementation. Next, we performed flow cytometric separation of candidate subpopulations as a means of segregating functional potential. These studies showed that CD45R/B220 expression levels appear to correlate with colony-forming ability. B-NHL cell fractions with lower levels of expression of the murine pan-B cell marker CD45R/B220 appear to be enriched for CFU activity in methylcellulose culture compared to B220-high cells. In addition, CD45R/B220-low B-NHL has higher tumorigenic potential (assessed by kinetics of growth rate) in assays of subcutaneous tumor nodule generation. These studies indicate differential potential of NHL subpopulations, and may begin to establish a phenotype of LySC. Finally, we measured in vitro clonogenic potential using two assays – i) limiting dilution suspension culture, and - ii) CFU assays. Interestingly, the results from these two independent assays do not correlate well in B-NHL; clonogenic potential appears to be substantially higher in limiting-dilution suspension cultures when compared to methylcellulose assays. These findings indicate that the relationship between CFU frequency and growth potential in B-NHL populations may differ from those found in normal hematopoietic cell types, and suggest that some caution should be exercised in the interpretation of B-NHL CFU assays.The adaptation of assays useful for identification of tumor-initiating sub-populations has also been useful in defining subsets of clonally related B-NHL cells that appear immunophenotypically and functionally distinct in vitro and in vivo. Our studies support the notion that B-NHL tumor-initiating cells may comprise a unique population amenable to prospective immunophenotypic and/or functional identification. Implicit in the concept of these putative LySC is that the control, and ultimately the eradication, of such unique NHL populations may result in improved clinical outcomes.[SAW and RMR contributed equally to this work. Funded by the Lymphoma SPORE, P50 CA130805501A1, PI: Richard Fisher, MD.] DisclosuresNo relevant conflicts of interest to declare.