WM Cells Research Articles

Pacritinib Blocks Key Pro-Survival Signaling Related to Mutated MYD88, Produces High Levels of Apoptosis and Overcomes Mutated BTK Cys481 Related BTK-Inhibitor Resistance

Background: Somatic mutations in MYD88 are prevalent in many B-cell malignancies, including Waldenström Macroglobulinemia (WM; 95-97%), primary CNS lymphoma (70-80%), ABC DLBCL (40%), marginal zone lymphoma (5-10%) and CLL (5-15%). Mutated MYD88 drives oncogenic pro-survival signaling through multiple signaling cascades that include HCK-directed BTK, ERK and SYK, as well as IRAK1/IRAK4 activation. HCK is an SRC family member that mutated MYD88 transcriptionally upregulates, and its activation is triggered by IL-6/IL-6R/gp130/JAK2 signaling. BTK inhibitors are active in MYD88 mutated B-cell malignancies, but fail to produce complete responses, and show varying durations of response that are disease dependent and impacted by depth of response. Acquired resistance to BTK inhibitors is commonly related to a BTK Cys481 mutation. Pacritinib is a kinase inhibitor, FDA-approved for myelofibrosis, that is well tolerated and targets pro-signaling cascades that are relevant to mutated MYD88 signaling, including IRAK1, JAK2, and SRC, a homologue of HCK ( Fig. 1). Pacritinib may therefore represent a more effective option in blocking mutated MYD88 pro-survival signaling versus BTK-inhibitors. Methods: We performed comparative studies to evaluate the activity of pacritinib and the covalent BTK-inhibitors ibrutinib and zanubrutinib on mutated MYD88 relevant pro-survival signaling, as well as proliferation and survival in MYD88 mutated cell lines and primary MYD88-mutated WM cells. Further to these studies, we assessed the activity of pacritinib in well-characterized mutant (BTK Cys481Ser) expressing covalent BTK-inhibitor resistant WM and ABC DLBCL lymphoma cell models. Results: Pacritinib produced higher levels of apoptosis in MYD88-mutated WM (BCWM.1) and ABC DLBCL (TMD-8), and primary (CD19 +) MYD88-mutated WM cells relative to the BTK inhibitors ibrutinib or zanubrutinib.The apoptotic activity of pacritinib showed dose dependence with high levels of apoptosis at 0.5 µM, which is well within its pharmacologically achievable levels. Importantly, pacritinib, in addition to its known effects on JAK2 and IRAK1 activation, also showed robust inhibition of p-HCK (Y411) and its downstream signaling partner p-BTK (Y223) in MYD88 mutated BCWM.1 and TMD-8 cells. By combination index and normalized isobologram analyses, pacritinib showed synergistic interactions with covalent BTK inhibitors, and more so with the BCL-2 inhibitor venetoclax in MYD88 mutated cell lines and primary WM cells. Lastly, pacritinib alone and combined with venetoclax induced high levels of apoptosis in BTK Cys481Ser expressing covalent BTK-inhibitor resistant MYD88 mutated WM and ABC DLBCL lymphoma cells. Conclusions: Pacritinib more broadly extinguishes mutated MYD88 pro-survival signaling cascades ( Fig 1.) and demonstrates high levels of apoptotic activityin mutated MYD88 WM and ABC DLBCL cells versus selective covalent BTK-inhibitors. Pacritinib also synergizes with covalent BTK- and BCL2 inhibitors and can overcome covalent BTK-inhibitor resistance related to mutated BTK Cys481. Our studies provide a framework for investigating pacritinib in MYD88 mutated lymphomas. A clinical trial of pacritinib in relapsed/refractory WM is being initiated.

Novel Protacs Demonstrate Dual Kinase Inhibition and Degradation of HCK and BTK, and Are Highly Active in MYD88 Mutated WM and ABC DLBCL Cells

Background: Activating mutations in MYD88 are prevalent in many B-cell malignancies, including Waldenström Macroglobulinemia (95-97%), primary CNS lymphoma (70-80%), ABC DLBCL (40%), marginal zone lymphoma (5-10%), and CLL (5-15%). Mutated MYD88 transcriptionally upregulates the SRC family member HCK, which in turn serves as a master signal for triggering multiple pro-survival cascades in mutated MYD88 lymphoma cells including BTK/NFKB, SYK, and ERK (Blood 127:3237-52; Blood Adv 4:141-153; Blood Adv 6:3332-38). In previous work, we developed and characterized a novel HCK kinase inhibitor, KIN-8194 (Blood 138:1966-79). KIN-8194 blocked both HCK and BTK and was active both in vitro and in vivo in MYD88 mutated xenograft lymphoma models, including BTK Cys481Ser ibrutinib resistant models. Proteolysis targeting chimeras (PROTACs) represent a novel approach for blocking kinase based signaling and may provide an advantage over kinase inhibitors with greater selectivity and sustained target inhibition. We therefore initiated a medicinal chemistry campaign to develop highly potent, selective, and bio-available HCK/BTK targeting PROTACS using KIN-8194 scaffold for the treatment of MYD88 driven lymphomas. Methods: We developed and characterized a large series of PROTACs using KIN-8194 as a scaffold. We rationally designed linkers to facilitate E3 ubiquitin ligase binding and improve physical properties leading to degraders with ideal oral pharmacokinetic properties and bioavailability. We characterized their kinome profiling by performing a KINOMEscan assay against a panel of 468 kinases at 1 uM ( Fig. 1). Subsequently, we conducted rigorous biological efficacy screenings using the ATP-based Cell Titer-Glo Luminescent Cell Viability Assay to identify the most potent compounds. Compounds with IC 50 scores lower than KIN-8194 were selected for further validation through flow cytometry based quantitative assays that verified apoptotic and cytotoxic effects on WM and MYD88-mutated lymphoma cell lines, as well as primary human peripheral blood mononuclear cells (PBMCs), utilizing Annexin V and Propidium iodide (PI) staining. Results: We identified and characterized several novel PROTACs from the KIN-8194 scaffold that exhibited potent dual inhibition of HCK and BTK kinase activity at 1 nM, as well as marked degradation of HCK and BTK by western blot analysis at dose levels as low as 100 nM in MYD88 mutated WM and ABC DLBCL cell lines. KINOMEscan profiling showed greater selectivity for novel PROTACs as exemplified in Fig. 1. Enhanced cell killing due to degradation of HCK and BTK versus inhibition alone was confirmed using respective PROTAC analogues with modified IMID warheads that do not bind cereblon. We observed suppression of downstream pro-survival NFKB and ERK signaling in response to the dual HCK/BTK PROTACs. Moreover, several HCK/BTK PROTACs (DFCI-004, -005, -006) demonstrated remarkable low IC 50 values (1-100 nM) in proliferation assays of MYD88 mutated BCWM.1 WM and TMD-8 ABC DLBCL cells. Importantly, the dual HCK/BTK PROTACs elicited higher levels of apoptosis in BCWM.1 WM and TMD-8 ABC DLBCL cells in comparison to the native KIN-8194 kinase inhibitor and showed sparing of healthy donor B- and T-cells akin to KIN-8194. Finally, murine PK studies showed that the HCK/BTK PROTACs DFCI-005 and -006 were highly bioavailable with F=40-42%, and C Max of 700-2700 nM at single oral dose ranges of 10-60 mg/kg. Conclusions: In these studies, we demonstrated the development and characterization of novel, dual HCK/BTK PROTACs that demonstrate potent and selective kinase inhibition and protein degradation of HCK and BTK. Importantly, the novel PROTACs showed enhanced apoptosis of MYD88 mutated WM and ABC DLBCL cells over the native kinase inhibitor KIN-8194 and sparing of healthy donor B- and T-cells. Lastly, novel HCK/BTK PROTACs exhibited high levels of bioavailability. Our studies therefore provide a framework for the advancement of dual HCK/BTK PROTACs for the treatment of MYD88 mutated lymphomas.

Pharmacokinetics-Pharmacodynamics Modeling and Evaluation of Tumor Response to Bortezomib Proteasome Inhibitor in Waldenstrom Macroglobulinemia.

Waldenstrom's macroglobulinemia (WM), also known as lymphoplasmacytic lymphoma, is a type of non-Hodgkin's lymphoma in which the malignant cells produce many macroglobulin proteins. It originates from B cells and develops in the bone marrow, where Wm cells combine to produce distinct types of blood cells, resulting in reduced volumes of red blood cells, white blood cells, and platelets, making it harder for the body to fight diseases. Chemoimmunotherapy is being used for the clinical management of WM, but new targeted agents, the BTK inhibitor ibrutinib and the proteasome inhibitor bortezomib, have shown significant improvements in patients with relapsed/refractory WM. However, given its effectiveness, drug resistance and relapse are normal, and there is little research on the pathways responsible for drug effects on the tumor. In this study, Pharmacokinetics-pharmacodynamic simulations were done to assess the effect of the proteasome inhibitor bortezomib on the tumor. For this purpose, the Pharmacokinetics-pharmacodynamic model was developed. The model parameters were determined and calculated using the Ordinary Differential Equation solver toolbox and the least-squares function. Pharmacokinetic profiles and pharmacodynamic analysis were performed to determine the change in tumor weight associated with the use of proteasome inhibitors. Bortezomib and ixazomib have been found to reduce tumor weight briefly, but once the dose is reduced, the tumor begins to grow again. Carfilzomib and oprozomib had better results, and rituximab reduced tumor weight more effectively. Once validated, it is proposed that a combination of selected drugs can be evaluated in the laboratory to treat WM.

Single-cell profiles reveal tumor cell heterogeneity and immunosuppressive microenvironment in Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is a rare and incurable indolent B-cell malignancy. The molecular pathogenesis and the role of immunosuppressive microenvironment in WM development are still incompletely understood. The multicellular ecosystem in bone marrow (BM) of WM were delineated by single-cell RNA-sequencing (scRNA-seq) and investigated the underlying molecular characteristics. Our data uncovered the heterogeneity of malignant cells in WM, and investigated the kinetic co-evolution of WM and immune cells, which played pivotal roles in disease development and progression. Two novel subpopulations of malignant cells, CD19+CD3+ and CD138+CD3+, co-expressing T-cell marker genes were identified at single-cell resolution. Pseudotime-ordered analysis elucidated that CD19+CD3+ malignant cells presented at an early stage of WM-B cell differentiation. Colony formation assay further identified that CD19+CD3+ malignant cells acted as potential WM precursors. Based on the findings of T cell marker aberrant expressed on WM tumor cells, we speculate the long-time activation of tumor antigen-induced immunosuppressive microenvironment that is involved in the pathogenesis of WM. Therefore, our study further investigated the possible molecular mechanism of immune cell dysfunction. A precursor exhausted CD8-T cells and functional deletion of NK cells were identified in WM, and CD47 would be a potential therapeutic target to reverse the dysfunction of immune cells. Our study facilitates further understanding of the biological heterogeneity of tumor cells and immunosuppressive microenvironment in WM. These data may have implications for the development of novel immunotherapies, such as targeting pre-exhausted CD8-T cells in WM.

Plasma cells arise from differentiation of clonal lymphocytes and secrete IgM in Waldenström macroglobulinemia

SummaryWaldenström macroglobulinemia (WM) is characterized by bone marrow infiltration with malignant lymphoplasmacytic cells (LPCs), a smaller population of plasma cells (PCs), and hypersecretion of IgM monoclonal protein. Here, we show that CD45low, CD38+, and CD138+ PCs and CD45high, CD38−, CD138-, CD19+, and CD20+ LPCs carry a heterozygous L265P mutation in the Toll-like receptor signaling adaptor MYD88. Both PCs and LPCs express the same auto-reactive IgHV sequences, suggesting a similar clonal origin and role for auto-antigens in WM cell survival. PCs are primarily responsible for IgM production even without substantial cell proliferation. When cultured in isolation, LPCs give rise to more differentiated PCs and secrete less IgM. Our analyses suggest that malignant PCs arise from the clonal LPC population, and are primarily responsible for IgM secretion in WM. Targeting malignant PCs may have therapeutic benefits in the treatment of WM and improve the duration of response and potentially, survival.

Abstract 6093: Mavorixafor enhances efficacy of Bruton tyrosine kinase inhibitors by overcoming the protective effect of bone marrow stroma on tumor cells in Waldenström’s macroglobulinemia

Abstract Waldenström’s macroglobulinemia (WM) is a rare B-cell malignancy characterized by monoclonal immunoglobulin M (IgM) hypersecretion and invasion of B cells in the bone marrow (BM) and lymphoid tissues. >90% of WM cases show mutations in MYD88 and 30-40% show mutations in the carboxyl terminus of CXCR4.The CXCR4/CXCL12 axis is crucial for the homing/retention of WM cells in the BM. Emerging clinical trial data (NCT04274738; ongoing) suggest that mavorixafor, a CXCR4 antagonist, in combination with ibrutinib results in clinically meaningful changes in levels of IgM and hemoglobin in patients with MYD88L265P CXCR4WHIM WM. However, the effects of mavorixafor with ibrutinib and other Bruton tyrosine kinase (BTK) inhibitors on WM cells harboring only the single mutation (MYD88L265P without CXCR4 mutation) have not been evaluated. This study was designed to test the ability of mavorixafor to sensitize WM cells carrying MYD88L265P CXCR4WT to BTK inhibitors in a WM/BM stromal cell (BMSC) co-culture model. The effects of mavorixafor on Ca2+ mobilization, cell migration, and adhesion to BMSC were also measured. WM cells (MWCL-1 cell line, MYD88L265PCXCR4WT) pretreated with mavorixafor and BTK inhibitors (ibrutinib, zanubrutinib, evobrutinib, LOXO-305, ARQ 531) were co-cultured with established BMSCs (HS27a cells). Cell viability, apoptosis, and IgM release were measured after 72 hours. BTK inhibitors, but not mavorixafor, decreased tumor cell viability and increased apoptosis of WM cells in the absence of BMSCs. Co-culture with BMSCs enhanced CXCR4 expression and protected WM cells from the effects of BTK inhibitors. BMSCs also significantly increased IgM secretion 2- to 5-fold compared with WM cells grown in the absence of BMSCs. Mavorixafor alone inhibited CXCL12-stimulated Ca2+ mobilization and migration of WM cells and disrupted the adhesion of WM cells to BMSCs. Combination of mavorixafor with BTK inhibitors overcame the protective effect of BMSCs on tumor cells, decreasing cell viability and/or increasing apoptosis compared with BTK inhibitors alone. Mavorixafor also reduced IgM secretion, which was further decreased when combined with BTK inhibitors.This study is the first to show in vitro that the protection of WM cells against BTK inhibitors conferred by BMSCs can be overcome by inhibition of the CXCR4/CXCL12 axis. The observations and responses to mavorixafor suggest a contribution of CXCR4WT to the pathogenicity of WM cells carrying only the MYD88L265P mutation. Mavorixafor addition enhanced the efficacy of not only ibrutinib but the other BTK inhibitors tested, supporting the greater potential of this combination therapeutic strategy in WM patients with or without CXCR4WHIM mutations. Further studies using additional WM cell lines and/or primary patient cells are warranted to support these findings. Citation Format: Chi Nguyen, Halenya Monticelli, Tom Kruitwagen, Matteo Tardelli, Barbara Maierhofer, Thalia Martins Rebelo, Sabine Maier-Munsa, Katarina Zmajkovicova, Lukas Dillinger, Adriana Badarau, Arthur G. Taveras. Mavorixafor enhances efficacy of Bruton tyrosine kinase inhibitors by overcoming the protective effect of bone marrow stroma on tumor cells in Waldenström’s macroglobulinemia [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 6093.

Waldenströms Macroglobulinemia Cell Lines Display Differential Surface Molecule Expression Reflecting Variability In Disease Biology and Potential Impact Of Therapeutic Stress

BackgroundWaldenstršms Macroglobulinemia (WM) is a lymphoplasmacytic lymphoma that is characterized by immunoglobulin-M (IgM) secreting B-cells that infiltrate the bone marrow, spleen and lymph nodes. The WM tumor compartment displays notable heterogeneity and is comprised of small lymphocytes, plasmacytoid lymphocytes and plasma cells in varying fractions that can be identified by flow cytometry. It has been recently shown that the WM cell profile can shift over time from predominantly monotypic B-lymphocytic cells to a more homogenous plasma cell population in response to treatment with various chemoimmunotherapeutics (Barakat et al, Am J Clin Pathol, 2011). This shift in cell populace is reflected by loss of characteristic B-lymphocyte surface antigens (CD19, CD20) and acquisition/overexpression of plasma cell markers (CD38, CD138), which can be detected by flow cytometry. WM is a relatively rare cancer and there is a corresponding scarcity of reliable preclinical models with only 3 molecularly validated human WM cell lines in existence. Although similar to one another, these human-IgM secreting models, namely BCWM.1 (developed from a newly diagnosed patient), MWCL-1 (developed from a relapsed/refractory patient) and RPCI-WM1 (developed in our laboratory from a highly refractory terminal WM patient) may represent distinct subclasses of WM. We investigated if the clinical variability at the time of development of these models had any phenotypic implications through altered expression of surface proteins. MethodsA comprehensive flow cytometric surface antigen analysis was performed using the BCWM.1, MWCL-1 and RPCI-WM1 cell lines. Surface antigens present on progenitor, immature, activated germinal center and memory B-cells along with those present on plasmablasts and plasma cells were examined. Presence of stem cell markers was also examined. Briefly, antibodies for CD5, 10, 11c, 13, 14, 16, 19, 20, 22, 23, 24, 25, 27, 28, 30, 32, 34, 35, 38, 39, 40, 43, 45, 45RA, 45RO, 52, 54, 56, 62L, 66b, 69, 70, 73, 79b, 80, 86, 90, 101, 105, 110, 111, 117, 123, 127, 133, 134, 135, 137, 138, 154, 184, 197, 202b, 243, 252, 268, 272, 278, 279, 309, 338, FMC7, HLADR, Kappa and Lambda were utilized. Antibodies for determining presence of intracellular CD247, 289, Kappa and Lambda were also used. ResultsWe observed notable differences between the immunophenotypic profiles of RPCI-WM1, BCWM.1 and MWCL-1. These variances are reflective of the dominating malignant B-cell population present at the time in the index patient from whom the cell lines were respectively established. By normalizing the mean fluorescent intensity signals to MESF values, we observed 14 differentially expressed antigens between BCWM.1 and MWCL-1, 23 CD antigens differentially altered between RPCI-WM1 vs. BCWM.1 and 29 surface markers differentially expressed in RPCI-WM1 relative to MWCL-1 cells (MESF fold change >2). Expression of 12 representative antigens across all WM models is shown in Table 1. [Display omitted] ConclusionThis is the first evaluation of the 3 most well established WM models. Our investigation displays important variations in surface protein expression and highlight biological distinction that may develop during the course of WM and/or exposure to therapeutics. This analysis validates that the RPCI-WM1 cell line was derived from a more advanced WM clone with predominantly plasmacytoid features that developed under ongoing therapeutic pressure and is consistent with the clinical phenotype of the index patient (rituximab, bortezomib and chemotherapy resistant). Understanding the significance of the distinct immunophenotypic profiles of existing WM models can (a) further improve the biology of WM, (b) impact of therapeutics on the WM clone and (c) engagement or disengagement of therapeutics targets (as is the case of CD20) during the disease course, which can help guide rational development of therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

Targeting Lymphoplasmacytic Lymphoma through a Novel Anti-FGF-Based Therapeutical Strategy

INTRODUCTION. Bone marrow (BM) angiogenesis plays a crucial role in several hematologic malignancies, including both myeloid neoplasms and lymphoproliferative disorders. Nevertheless, the contribution of angiogenesis in patients with lymphoplasmacytic lymphoma/Waldenstrom's Macroglobulinemia (WM) and how it might be targeted for potential therapeutical intervention has not been described.METHODS. Publically available mRNA profiling datasets of primary bone marrow (BM)-derived CD19 WM cells were used (i.e. GSE6691; GSE9656). The Gene Set Enrichment Analysis (GSEA) computational method was adopted (FDR<0.25; P<0.05). Co-culture proliferation and adhesion assays were performed using available WM (BCWM.1; MWCL1; WMWSU), IgM secreting cell lines (MEC1), and primary WM bone marrow (BM)-derived mesenchymal stromal cells (MSCs). Cell viability of primary CD19 positive-selected BM WM cells and peripheral blood-derived mononuclear cells (PBMC) was assessed by MTS assay. Modulation of signaling pathways was studied at protein level using western blot analysis. A pan-FGF trap NSC12 molecule was identified by a virtual screening of a NCI small molecule library: it acts a trap able to block the FGF2/FGFR interaction. NSC12 was tested in order to define its potential anti-WM activity in vitro.RESULTS. GSE6691 gene expression dataset was used to investigate the most differentially expressed mRNA in WM cells as compared to their normal cellular counterpart: the top two pathways (ranked based on the normalized enrichment score and p-value) were related to “TNF-α signaling via NF-kB” and “angiogenesis”. It has been already reported on the constitutive activation of NF-kB in WM; and therefore selected the “angiogenesis cascade” for further investigation (FDR 0.10; P 0.03). FGFR-1 was one of the most up-regulated genes in WM cells. We therefore looked at FGF- and FGFR-related mRNAs, using an independent GEP data set (GSE9656); and found FGFR-1, FGF-2, -12, -17 and -18 to be significantly enriched in WM cells as compared to their normal cellular counterpart (P<0.05). We next sought to determine the functional sequelae induced by the NSC12-mediated FGF2/FGFR blockade on WM cells. NSC12 exerted a direct anti-WM activity against all the cell lines tested (IC50: 3mM; P <0.05). Importantly, NCS12-induced cytotoxicity was demonstrated using WM patients' BM-derived CD19+ cells. NSC12 selectively targeted the FGF/FGFR axis, as demonstrated by inhibition of phospho(p)-FGFR1, as well as of downstream targets such as p-FSR2, p-JAK2, p-STAT3, and p-ERK. Of note, NSC12 inhibited WM cell proliferation even in a co-culture system with primary WM BM-MSCs. BM-MSC-induced up-regulation of p-FGFR1, p-STAT3, p-AKT was inhibited in WM cells exposed to increasing NCS12 doses, thus suggesting the activity of the compound even in the presence of the supportive BM milieu. We further confirmed NSC12-dependent induction of pro-apoptotic pathways, as shown at protein level by enhanced caspase-3- and PARP-cleavage. Notably, NSC12 did not induce cytotoxicity against healthy donor-PBMC.CONCLUSIONS. These findings provide novel insights into the pathogenesis of WM, describing the up-regulation of the FGF/FGFR axis in WM cells, thus providing the preclinical rational for targeting FGF in this disease. Consistently, the novel pan-FGF trap NSC12 demonstrated an anti-WM activity, through both a direct and an indirect effect against WM cells and the WM cell/BM interaction, respectively. DisclosuresRossi:Amgen: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

Aberrant HCK Survival Signaling By Mutated MYD88 Requires PAX5 in Waldenstrom's Macroglobulinemia and ABC DLBCL Cells

Activating mutations in MYD88 are present in 95% and 40% of patients with WM and ABC DLBCL, respectively. MYD88 mutations are known to trigger growth and survival of WM and ABC DLBCL cells through BTK/IRAK1/IRAK4 directed NF-kB signaling (Ngo et al, Nature 2011; Yang et al, Blood 2013). Recently, we identified that mutated MYD88 can trigger the SRC family member HCK, a kinase that is downregulated in later stages of B-cell ontogeny (Yang et al, Blood 2016). HCK promotes malignant cell survival through activation of BTK, as well as AKT and ERK1/2, whereas knockdown of HCK leads to decreased survival in WM and ABC DLBCL cells. The regulatory mechanisms that promote mutated MYD88 directed HCK signaling are unknown, and may permit therapeutic advances for MYD88 mutated diseases. To clarify whether HCK expression is regulated by TLRs / MYD88 or BCR signaling, we performed activation studies using triggers of TLR9 (ODN-2006) and TLR4 (LPS-EP) signaling, and BCR (anti-IgM/IgG) signaling in MYD88 wild-type or mutated B-cell lymphoma cells. Stimulation of TLRs but not BCR increased HCK transcription, particularly in wild-type MYD88 expressing B-cell lymphoma cells thereby demonstrating the importance of TLR/MYD88 signaling in driving HCK expression. To better understand the mechanism for the aberrant upregulation of HCK transcription in MYD88 mutated B-cell lymphoma cells, we performed promoter-binding TFs profiling, and observed that among the TFs, PAX5 signal was reduced the most by the addition of HCK promoter into BCWM.1 cell nuclear extracts. The signals of many other TFs that can be regulated by NF-kB (AP1, C\\EBP, Myb, NF-kB), MAPK (AP1, C\\EBP, CREB, HNF4, NF-1) and STAT3 signaling were also reduced. These results indicate that PAX5 and mutated MYD88 directed TFs including NF-kB, AP-1 and STAT3 bind to endogenous HCK promoter sequence. HCK promoter binding consensus sequence analysis and ChIP studies confirmed the presence of PAX5, as well as AP-1, NF-kB and STAT3 binding sites on the HCK promoter. Knockdown of PAX5 reduced HCK mRNA level in BCWM.1 and TMD-8 cells indicating an activating function of PAX5 in the regulation of HCK expression. The deletion of AP-1, NF-kB and STAT3 binding sites on HCK promoter reduced HCK promoter activity, thereby supporting a role for AP-1, NF-kB and STAT3 in HCK gene expression in MYD88 mutated cells. Treatment of BCWM.1 and TMD-8 cells with inhibitors of AP-1, NF-kB and STAT3 decreased HCK promoter activities by luciferase reporter assay, as well as endogenous HCK mRNA transcription levels in MYD88 mutated cells. Our data indicate PAX5 along with mutated MYD88 triggered transcription factors NF-kB, AP-1 and STAT3 orchestrate aberrant HCK expression. The findings provide a framework for efforts directed at blocking MYD88 triggered HCK survival signaling in MYD88 mutated B-cell lymphomas. DisclosuresCastillo:Abbvie: Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.

Beyond MYD88: Implications of TNFAIP3 Loss on MYD88 Driven Lymphomas

MYD88 mutations are one of the most recurrent mutations in hematologic malignancies and are found in 19% of lymphomas. While it is detected in many subtypes of B-cell malignancies, its prevalence is highest in Waldenstrom Macroglobulinemia (90-100%, WM) and the ABC subtype of diffuse large B-cell lymphoma (39%, DLBCL). While the biologic impact of MYD88L265P has been well-studied, little is known about its function in the context of a complex genetic background, which is often seen in DLBCL. Recent mouse models suggest that MYD88L265P alone may not be sufficient to induce tumor formation and that the pathway requires additional genetic hits. Interplay between MYD88L265P and other oncogenic events are further supported by the fact that TNFAIP3 (A20) inactivation often accompanies MYD88L265P . This data suggests a potential relationship between these genetic events, and more importantly, a possible clinical impact. The aim of this study was to determine the clinical impact of MYD88L265P and TNFAIP3 loss and to generate a human cell line model that mimics the effect of MYD88L265P / TNFAIP3 loss in hopes to understand the cellular consequence of these genomic alterations and identify therapeutic targets.MYD88 mutations have been reported to associate with a poor outcome in DLBCL and upon review of our whole exome sequencing and clinical data (n=147) we found that mutations in MY D88 (n=20) are commonly found in combination with TNFAIP3 loss (n=11, 55%). Additionally, review of RNASeq data (n=33) reveals that the MYD88 and NF-κB target genes IL-6 and BCL2 are elevated (1.4 fold and 1.5 fold) in patients with TNFAIP3 loss (n=7). To better understand the impact of a TNFAIP3 loss in combination with the MYD88L265P mutation,we used the WM cell line MWCL1 and the ABC-DLBCL cell line HBL-1, both of which harbor a heterozygous mutation in MYD88L265P . For the TNFAIP3 specific knock out, we used the transcription activator-like effector nucleases (TALENs) genome editing approach to induce a double stranded break in exon 5 of the TNFAIP3 gene. TALENs were expressed in lymphoma cells by transient transfection, and single-cell clones were isolated. Sanger sequencing of the TNFAIP3 gene was performed to identify clones that carried the deletion and loss of A20 was confirmed by Western blot. We then explored the impact of TNFAIP3 loss on MYD88L265P-driven p38 and NF-κB activation and found that phosphorylation of p38 and NF-κB is significantly higher in the MWCL1 and HBL-1 A20 knockout cell lines (p≤ 0.05). We next looked at expression of NF-κB target genes by quantitative real time PCR and found that IL-6 and CXCL-10 (IP-10) were significantly upregulated in the MWCL1 and HBL-1 A20 knockout cell lines compared to their wild type cell lines (p≤ 0.05). Upregulation of IL-6 and CXCL-10 protein expression was confirmed by Luminex. Because of the increased cytokine expression detected in A20 knockout cells, we next looked at JAK/STAT signaling proteins and found that phosphorylation of STAT3 was higher in MWCL1 and HBL-1 A20 knockout cells.In summary, we have established new WM and DLBCL cell line models that mimic the effect of the MYD88L265P mutation in combination with a loss of the TNFAIP3 gene and A20 expression. We show that loss of TNFAIP3 results in a higher baseline phosphorylation of NF-κB, p38 and STAT3. Additionally, loss of TNFAIP3 impacts expression of IL-6 and IP-10. Overall, results from this study contribute to our understanding of MYD88 driven lymphomas, suggests a possible clinical implication for those individuals that harbor both a MYD88 mutation and a loss of TNFAIP3, and also provides us with a useful model to study novel therapeutic strategies in patients who harbor these genetic variants. DisclosuresWitzig:Kura: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Novartis: Research Funding. Ansell:Seattle Genetics: Research Funding; Celldex: Research Funding; Bristol-Myers Squibb: Research Funding; Merck: Research Funding; Affimed: Research Funding. Cerhan:Janssen: Other: Scientific Advisory Board (REMICADELYM4001); Janssen: Other: Multiple Myeloma Registry Steering .

Waldenström’s Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells

Waldenström’s Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells

A Retrospective Study of 67 Inflammatory Waldenström’s Macroglobulinemia

Introduction Waldenström's macroglobulinemia (WM) is an indolent lymphoma with medullary infiltration by a lymphoplasmacytic clone associated with an immunoglobulin M paraprotein. Some patients diagnosed with WM present a concomitant inflammatory syndrome: increased C-reactive protein (CRP) levels, fever, recurrent night sweats, anorexia. These patients were usually referred as inflammatory WM despite no definition has been established. Here, we describe clinical and biological characteristics of inflammatory WM patients, response to treatment, survival and discuss pathogenesis hypotheses. Methods In this descriptive retrospective study, we included WM patients followed at Saint-Louis hospital between 2007 and 2019 with WM diagnosis fulfilling the World Health Organization's criteria, with medullary infiltration proven by bone marrow biopsy or aspiration examination and monoclonal M paraprotein, associated with a biological inflammatory syndrome with or without clinical inflammatory signs. Inflammatory WM diagnosis was retained in the absence of differential diagnoses for the inflammatory syndrome such as cancer, infection, autoimmune or inflammatory diseases, using complete blood workup, PET and body scanner. Results Two-hundred and forty-two patients were included: 67 of inflammatory phenotype (28%), 166 non-inflammatory (68%) and 9 with inflammatory syndrome of unknown origin (4%). Describing inflammatory WM, diagnoses were made between 1985 and 2018, 50% after 2007. At treatment initiation, median age was 69 years (range 45-94), monoclonal IgM median value was 24.9 g/L (4.8-77.3) (Table 1). Median CRP was 40.5mg/L (IQR 23.8-64.8). CRP was correlated with fibrinogen (r =0.53, 95% confidence interval (CI) 0.18-0.76, p=0.006) and inversely correlated with albuminemia (r=-0.65, CI -0.82- -0.39, p &amp;lt; 0.001). Mean medullary infiltration was 50%. Three of 35 patients with avalaible karyotype had 6q deletion. Only 12 patients had medullary molecular analysis: 9 of them harboured MyD88 L265P mutation, 2 p53 mutations and none CXCR4 WHIM mutation. Five cryoglobulin tests were positive, Coombs test positive for 3 patients and anti-MAG antibodies positive for 2. Indications for front-line therapy were cytopenias in 44 patients (88%), inflammatory syndrome for 20 (40%), systemic symptoms in 15 (30%), tumoral syndrome in 10 (20%) and hyperviscosity syndrome in 3 (6%). Sixty-two patients were treated, at front-line 60% received rituximab-based therapy and 39% monotherapy. Overall response rate was 84%. Inflammatory and hematological response were statistically correlated (OR 13.8, CI 2.07-74.6, p = 0.005). Median follow-up was 12.6 years (IQR 7.0-22.8). Median progression-free survival (PFS) after front-line therapy was 32 months and estimated PFS was 68% (CI 55-78) at 10 months, 59% (CI 46-70) at 20 months and 30% (CI 19-42) at 5 years. Overall survival (OS) for inflammatory and non-inflammatory WM patients were not significantly different (median OS 14.0 and 16.4 years, respectively, c2 0,14, p=0,71, Figure 1). At 5 years, estimated OS was 85% (CI 71-93) inflammatory patients and 84% (CI 75-90) for non-inflammatory, at 10 years 66% (CI 48-79) and 70% (CI 58-79). Three patients were secondarily diagnosed with diffuse large B-cell lymphoma (DLBCL). Conclusions We reported 67 WM patients with inflammatory phenotypes, representing a third of WM patients in our cohort. Compared with literature, their clinical and biological characteristics do not differ from non-inflammatory WM patients, as well as response to treatment and PFS. There is no excess of transformation into DLBCL. Our inflammatory and non-inflammatory cohorts show no difference in terms of OS. Inflammatory response is strongly correlated with hematological response. Non-decrease of CRP level during treatment and increase of CRP during follow-up in inflammatory WM patients should be a warning sign for refractory disease or relapse. Further studies are needed to better delineate this sub-group of patients and explore its pathogenesis. Studying medullary cytokinic environment, WM cells, tumoral microenvironment and M paraprotein contributions could elucidate the underlying pathophysiological mechanisms involved. Disclosures Thieblemont: Roche, Hospita: Research Funding; Roche, Amgen, Kyte Gilead, Celgene, Abbvie, Novartis, Cellectis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Cellectis: Speakers Bureau.

Novel tumor-targeted liposomes comprised of an MDM2 antagonist plus proteasome inhibitor display anti-tumor activity in a xenograft model of bortezomib-resistant Waldenstrom macroglobulinemia

Targeted drug delivery remains an active area of investigation in hematologic cancers. We have previously reported on a novel nanoparticle formulation (D1X) that can encapsulate drugs within a liposome whose lipid bilayer contains dexamethasone, which serves as a targeting ligand for drug delivery to tumor cells that express glucocorticoid receptors. We tested the activity of D1X-encapsulated bortezomib (D1XB) in combination with D1X-encapsulated nutlin (D1XN) in B-lymphoma/Waldenstrom macroglobulinemia (WM) cells. WM cells treated with D1XB + D1XN experienced cell cycle arrest, ER stress and apoptosis. In mice xenografted with bortezomib-resistant WM cells, D1XB + D1XN treatment resulted in significantly lower tumor burden compared to vehicle or nonliposomal parent drugs. In vivo biodistribution studies showed minimal uptake of D1X-based drugs in normal mice tissues. Our studies demonstrate that highly targeted delivery of both bortezomib and nutlin encapsulated in D1X nanoparticles are cytotoxic to and delay in vivo growth of bortezomib-resistant WM cells.

SYK is activated by mutated MYD88 and drives pro-survival signaling in MYD88 driven B-cell lymphomas

Activating MYD88 mutations promote pro-survival signaling through BTK and HCK, both targets of ibrutinib. Despite high response rates, complete responses to ibrutinib are lacking, and other MYD88 triggered pro-survival pathways may contribute to primary drug resistance. B-cell receptor (BCR) signaling has been observed in lymphomas driven by mutated MYD88, even without activating the BCR pathway mutations. We identified activated SYK (p-SYK), a component of BCR in complex with MYD88 in MYD88-mutated WM and ABC DLBCL lymphoma cells. Confocal microscopy confirmed co-localization of MYD88 with SYK in MYD88-mutated cells. Knockdown of MYD88 or use of a MYD88 signaling inhibitor abrogated SYK activation, while expression of mutated but not wild-type MYD88 amplified p-SYK in MYD88-mutated and wild-type lymphoma cells. Knockdown of SYK or use of inhibitors targeting SYK blocked p-STAT3 and p-AKT signaling in MYD88-mutated cells. Cell viability analysis showed that combining ibrutinib and SYK inhibitors triggered synthetic killing of MYD88-mutated lymphoma cells. Our findings extend the spectrum of mutated MYD88 pro-survival signaling to include SYK directed BCR cross talk in MYD88-mutated lymphomas. Targeting SYK in combination with ibrutinib produces synthetic lethality, providing a framework for the clinical investigation of ibrutinib with SYK inhibitors in MYD88-mutated lymphomas.

Mutated MYD88 Regulates HCK Pro-Survival Signaling through JunB in MYD88 Mutated B-Lymphoma Cells

Hematopoietic cell kinase (HCK) is a member of the SRC family tyrosine kinases (SFKs) that is down-regulated in later stages of B-cell ontogeny. In MYD88 mutated B-cell lymphomas, HCK is aberrantly over-expressed and is activated and triggers multiple growth and survival pathways including BTK, PI3Kδ/AKT and ERK1/2 which are essential to tumor cell survival. Ibrutinib, a pleiotropic inhibitor of BTK, that produces remarkable responses in MYD88 mutated WM, ABC-DLBCL, and Primary CNS Lymphoma was found also potently inhibits HCK. Mutations that abolish ibrutinib-HCK binding greatly diminish anti-tumor activity in MYD88 mutated lymphoma cells, highlighting the importance of HCK as an essential target in MYD88 driven diseases. To clarify the transcriptional regulation of HCK in MYD88 mutated malignancies, we performed promoter binding TF profiling, PROMO weighted transcription factor (TF) consensus binding analysis, and chromatin immuno-precipitation (ChIP) studies. We identified PAX5, and mutated MYD88 downstream signaling mediators, STAT3, AP-1 and NF-kB as important drivers of HCK transcription. Knockdown of PAX5, a crucial regulatory factor required for B-cell commitment and identity, abrogated HCK transcription in MYD88 mutated lymphoma cells. Deletion of STAT3, AP-1 or NF-kB binding sites greatly reduced corresponding TFs binding and HCK promoter activity, indicating the importance of MYD88 directed signaling in the regulation of HCK transcription. Among AP-1 complex components, JunB but not c-Jun showed greatest relevance to TLR/MYD88 signaling and regulation of HCK transcription. Since STAT3 and NF-kB are known downstream mediators of mutated MYD88 signaling, we focused on the function of JunB. JunB phosphorylation increased following MYD88 pathway activation through TLR4 (with LPS-EB) or TLR9 (with ODN-2006), as well as lentiviral mediated expression of mutated MYD88 (L265P) but not wild type MYD88 (MYD88-WT) at Thr102 and Thr104 in either MYD88 mutated BCWM.1 cells or MYD88-WT Ramos cells. Conversely, c-Jun phosphorylation was not impacted by either intervention. Knockdown of MYD88 in BCWM.1 WM cells also reduced the phosphorylation of JunB, while c-Jun phosphorylation showed modest increase. Moreover, knockdown of JunB reduced HCK protein levels in MYD88 mutated WM and ABC-DLBCL cells. These data demonstrate that JunB is an important mediator of mutated MYD88 signaling among AP1 complex members and is directly involved in the regulation of HCK expression in MYD88 mutated B-cell lymphoma. The findings provide new insights into the transcriptional regulation of HCK by MYD88 driven TFs, and opportunities for further advancing targeted therapeutics in MYD88 driven B-cell malignancies. Disclosures Hunter: Janssen: Consultancy. Castillo:TG Therapeutics: Research Funding; Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Research Funding; BMS: Research Funding; Janssen: Consultancy.

A Novel HCK and BTK Dual Inhibitor Kin-8194 Shows Superior Activity over Ibrutinib and Overcomes BTKC481S Mediated Ibrutinib Resistance in Vitro and In Vivo in MYD88 Mutated B-Cell Lymphomas

Activating mutations in MYD88 promote malignant cell growth and survival through multiple pathways that include BTK and HCK. HCK is transcriptionally upregulated and activated by mutated MYD88 and in turn activates BTK itself, as well as ERK and AKT. Ibrutinib is a covalent inhibitor that binds to BTKCys481 and shows activity in MYD88 mutated B-cell malignancies, including WM, MZL, ABC DLBCL, and PCNSL. Resistance to ibrutinib on the basis of BTKCys481 as well as downstream mutations is increasingly being recognized. We therefore sought to develop potent and selective inhibitors that target HCK. We developed a non-covalent dual inhibitor, KIN-8194, of HCK and BTK following a screen &amp;gt;220 clinical and preclinical kinase inhibitors, and lead optimization following synthesis &amp;gt;400 analogs. Dual HCK and BTK inhibition was confirmed by both KINOMEscan® and live-cell target engagement studies (KiNativ™ profiling and live cell ATP-biotin competition assay). KIN-8194 showed robust suppression of HCK (IC50&amp;lt;0.495nM) and BTK (IC50=0.915nM) in these studies, and blocked pHCK and pBTK in both wild-type and mutated BTKCys481 WM and ABC DLBCL cells, and primary WM cells. Importantly, KIN-8194 showed selective killing of MYD88 mutated WM and ABC DLBCL cells and overcame resistance to ibrutinib in WM and ABC DLBCL cells engineered to express mutated BTKCys481. KIN-8194 showed excellent microsomal stability across multiple species including human (T1/2=49.5 minutes). Pharmacokinetic studies in mice showed excellent bioavailability (F=55%), serum half-life (T1/2=15.1 hours) and drug clearance (CL=17.4mL/min/kg) amenable to once daily oral dosing. The compounds exhibited an excellent in vitro safety profiling including no relevant inhibition observed against a panel of 100 other receptor targets, including hERG, AMES was negative up to 50 µM, and Cyp inhibition studies showed acceptable inhibition up to 10 µM. Pharmacodynamic studies following oral administration showed that KIN-8194 blocked both pHCK and pBTK in wild-type and mutated BTKCys481 expressing TMD8 ABC DLBCL cells engrafted in NOD SCID mice. Continuous dosing up to 100 mg/kg was well tolerated in these mice. KIN-8194 treated NOD SCID mice xenografted with either wild-type (A) or mutated (B) BTKCys481 TMD cells (N=8/cohort) showed superior tumor growth suppression and survival over vehicle control or ibrutinib treated mice at 50 mg/kg. Among wild-type BTKCys481 TMD8 xenografted mice treated for 6 weeks, elimination of tumor was observed in half the mice with no subsequent growth following 12 additional weeks of observation, consistent with a cure. We therefore describe a novel, highly potent non-covalent dual HCK and BTK inhibitor that is well tolerated in mice, shows selective killing of MYD88 mutated WM and ABC DLBCL cells, and can overcome mutated BTKCys481 related ibrutinib resistance. Disclosures Hunter: Janssen: Consultancy. Castillo:Beigene: Consultancy, Research Funding; TG Therapeutics: Research Funding; Abbvie: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Gray:Gatekeeper, Syros, Petra, C4, B2S and Soltego.: Equity Ownership; Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield and Sanofi.: Equity Ownership, Research Funding. Treon:Pharmacyclics: Research Funding; BMS: Research Funding; Janssen: Consultancy.

Waldenström's Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells

Waldenström's Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells

Dual PAK4-NAMPT Inhibition Impacts Growth and Survival, and Increases Sensitivity to DNA-Damaging Agents in Waldenström Macroglobulinemia.

p21-activated kinase 4 (PAK4) plays a significant biological and functional role in a number of malignancies, including multiple myeloma (MM). On the basis of our promising findings in MM, we here characterize PAK4 expression and role in WM cells, as well effect of dual PAK4-NAMPT inhibitor (KPT-9274) against WM cell growth and viability. We have analyzed mRNA and protein expression levels of PAK4 in WM cells, and used loss-of-function approach to investigate its contribution to WM cell viability. We have further tested the in vitro and in vivo effect of KPT-9274 against WM cell growth and viability. We report here high-level expression and functional role of PAK4 in WM, as demonstrated by shRNA-mediated knockdown; and significant impact of KPT-9274 on WM cell growth and viability. The growth inhibitory effect of KPT-9274 was associated with decreased PAK4 expression and NAMPT activity, as well as induction of apoptosis. Interestingly, in WM cell lines treated with KPT-9274, we detected a significant impact on DNA damage and repair genes. Moreover, we observed that apart from inducing DNA damage, KPT-9274 specifically decreased RAD51 and the double-strand break repair by the homologous recombination pathway. As a result, when combined with a DNA alkylating agents bendamustine and melphalan, KPT-9274 provided a synergistic inhibition of cell viability in WM cell lines and primary patient WM cells in vitro and in vivo. These results support the clinical investigation of KPT-9274 in combination with DNA-damaging agent for treatment of WM.

MYD88 Triggered SYK Activation Promotes BCR Cross-Talk, and Identifies SYK As a Novel Therapeutic Target of Mutated MYD88 Signaling

Activating mutations in MYD88, a component of the Toll-receptor (TLR) pathway, trigger Myddosome self-assembly and multiple downstream pro-survival signaling through BTK and IRAK4/IRAK1 (Yang et al, Blood 2013). Activation of B-cell receptor (BCR) signaling has also been observed in WM, though the mechanism(s) remain to be clarified (Argyropoulos et al, Leukemia 2016). While activating mutations in the BCR components CD79A/B are common in MYD88 mutated ABC DLBCL, they are uncommon in WM (Ngo et al, Nature 2011; Hunter et al, Blood 2014). We therefore sought to clarify if TLR/MYD88 crosstalk could explain aberrant BCR signaling in WM. We performed PhosFlow analysis of MYD88 and BCR signaling components of MYD88 mutated and wild-type WM and ABC DLBCL cell lines. These studies showed high levels for expression of the BCR component p-SYK (Y525-526) in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1) cell lines versus MYD88 wild-type cell lines. High levels of p-SYK were also observed in primary MYD88 mutated WM cells compared to healthy donor peripheral blood B-cells. Following treatment with a MYD88 blocking peptide, p-SYK was robustly reduced in WM cell lines, while only modest reduction was observed in ABC DLBCL cell lines which also carry activating CD79B mutations. Use of MYD88 inhibitor also blocked the p-SYK in primary MYD88 mutated WM cells. Lentiviral over-expression of MYD88 L265P but not wild-type MYD88 or vector control augmented p-SYK expression in MYD88 mutated BCWM.1 WM cells, as well as MYD88 wild-type Ramos and OCI-Ly7 cells. Conversely, knockdown of MYD88 in BCWM.1 cells confirmed the dependence of p-SYK on mutated MYD88. p-SYK was also confirmed to be in complex with the Myddosome in BCWM.1 cells by co-immunoprecipitation (co-IP) using anti-MYD88 and anti-SYK/anti-p-SYK specific antibodies. Over-expression and knockdown studies of mutated MYD88 also showed an association of downstream p-STAT3 (Y705) signaling on MYD88 triggered p-SYK, a finding confirmed by use of SYK inhibitors which blocked p-STAT3 in a dose-dependent manner. CellTiter-Glo® viability assays showed the SYK inhibitors, R406 and Entospletinib (GS-9973), produced higher cytotoxicity in MYD88 mutated versus wild-type B-cell lines. (Figure 1A) Since BCR/SYK kinase triggers divergent signaling from TLR/BTK/IRAK pathways, we examined the combined effect of BTK and SYK inhibition. Combination index and normalized isobologram analysis demonstrated synergistic effects with the combination of ibrutinib with either R406 or Entospletinib in MYD88 mutated WM and ABC DLBCL cell lines (Figure 1B, 1C). The combination of ibrutinib with either R406 or Entospletinib also produced more robust tumor cell apoptosis in primary MYD88 mutated WM cells. Our findings show that MYD88 can cross-talk with BCR pathway through SYK tyrosine kinase, and trigger aberrant p-STAT3 signaling. The inhibition of both TLR/BTK and BCR/SYK activation by use of ibrutinib and SYK inhibitors produced synergistic tumor cell killing of MYD88 mutated WM and ABC DLBCL lymphomas, and provides a framework for clinical studies aimed at extinguishing aberrant MYD88 signaling. [Display omitted] DisclosuresCastillo:Millennium: Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Genentech: Consultancy; Beigene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding. Hunter:Pharmacyclics: Consultancy. Gray:Syros, Soltego, Petra, C4 Therapeutics: Equity Ownership. Treon:Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Research Funding; Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses.

A Novel HCK Inhibitor Kin-8193 Blocks BTK Activity in BTKCys481 Mutated Ibrutinib Resistant B-Cell Lymphomas Driven By Mutated MYD88

Activating mutations in MYD88 alone or in coordination with BCR activating mutations transactivate Bruton's tyrosine kinase (BTK) in WM and ABC DLBCL cells (Yang et al, Blood 2013; Wilson WH et al, Nat Med 2015; Phelan JD, Nature 2018). Ibrutinib is a covalent inhibitor that binds to BTKCys481 and is active in MYD88 mutated WM and ABC-DLBCL (Treon et al, NEJM 2015; Wilson et al, Nat Med, 2015). Acquired resistance to ibrutinib is increasingly being recognized in WM, as well as other B-cell malignancies due to somatic mutations at BTKCys481 that abrogate BTK-ibrutinib binding (Xu et al, Blood 2017). BTKCys481 mutations are usually sub-clonal, but can drive ibrutinib resistance and protect BTK wild-type clones through a paracrine mechanism involving activation of ERK1/2 (Chen et al, Blood 2018). Hematopoietic cell kinase (HCK) which is aberrantly upregulated and transactivated by mutated MYD88, and triggers multiple pro-survival signaling cascades that include activation of BTK, as well as PI3K/AKT and MAPK/ERK1/2 (Yang G et al, Blood 2016). We therefore examined if inhibition of HCK could abrogate BTK activity and overcome ibrutinib resistance driven by BTKCys481 mutations in MYD88 mutated B-cell lymphomas. We performed a screen of 220 clinical and preclinical kinase inhibitors to identify compounds with potent HCK inhibition. Over 100 analogs of three series of promising compounds with HCK activity were synthesized and triaged. Target deconvolution was performed to clarify selectivity, and other important kinase targets. These efforts led to the selection of a lead compound identified as a type-II kinase inhibitor, KIN-8193, with a molecular weight around 600. Single-digit nanomolar (nM) biochemical and double-digit nM cellular potency was demonstrated, with high selectivity (S-score 0.07) in line with that observed with ibrutinib (S-score of 0.03). Live-cell target engagement for HCK was confirmed by competitive ATP-biotin binding assay. DMPK and PK studies showed very high levels of mouse, rat, and human microsomal stability (42.4, 60.2 and 79.3 minutes respectively), and oral bioavailability in mice (48%) and rats (79%). Cmax reached 2.0 µM, while T1/2 was 26.8 hours with 25 mg/kg single oral dosing in rats. Rats toxicology studies showed excellent tolerability in 28-days repeated oral dosing with 25 mg/kg/biw dosing schedule. No relevant inhibition was observed against a panel of 100 other receptor targets, including hERG. AMES was negative up to 100 uM, and Cyp inhibition studies showed acceptable inhibition up to 10 uM. KIN-8193 potently inhibited the phosphorylation of HCK(p-Y411) and its downstream target BTK(p-Y223) in both BTK wild-type and ibrutinib-resistant BTKCys481 mutated WM and ABC DLBCL cell lines driven by activating MYD88 mutations, and primary WM cells (Fig 1A). Target engagement studies showed HCK but not BTK direct binding. In vivo pharmacodynamic (PD) studies using luciferized BCWM.1 cells WM xenograft mouse model showed potent dose-dependent inhibition of HCK(p-Y411) and BTK(p-Y223) at 6 and 24 hours following single dose administration of KIN-8193. Importantly, KIN-8193 showed selective cytotoxicity against MYD88 mutated BTK wild-type and BTKCys481 mutated, ibrutinib-resistant WM and ABC DLBCL cell lines, and primary WM cells, but had no impact on healthy donor B- and T-cells at pharmacologically achievable levels (Fig. 1B). We therefore describe a novel, highly selective and potent HCK inhibitor that is well tolerated in long-term rat toxicology studies and shows selective killing of MYD88 mutated WM and ABC DLBCL cells. Inhibition of HCK by KIN-8193 blocks downstream wild-type BTK and Cys481 mutated BTK activity, and overcomes ibrutinib resistance induced by BTKCys481 mutations. [Display omitted] DisclosuresHunter:Pharmacyclics: Consultancy. Castillo:Genentech: Consultancy; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Millennium: Research Funding. Gray:Syros, Soltego, Petra, C4 Therapeutics: Equity Ownership. Treon:Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Research Funding; Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses.