Activated B-cell Diffuse Large B-cell Lymphoma Research Articles

Abstract PO-036: Targeting the CBM signalosome with a MALT1 scaffolding inhibitor for treatment of non-Hodgkin lymphomas

Abstract Introduction: The CBM signalosome (CARDs/BCL10/MALT1) is an evolutionarily conserved key regulator of canonical NF-κB signaling. Activating mutations of the CBM signalosome constitutively activate NF-κB, driving tumorigenesis in B-cell Non-Hodgkin Lymphoma (B-NHL). Depletion of individual components of the CBM complex including MALT1 was shown preclinically to suppress NF-κB driven Activated B-Cell Diffuse Large B-Cell Lymphoma (ABC-DLBCL), inspiring many efforts to develop MALT1 inhibitors for therapeutic use. MALT1 is a dual function protein with two independent roles. Primarily, MALT1 is a scaffold protein organizing the CBM complex and directly transducing canonical NF-κB signaling. Secondarily, MALT1 is a paracaspase, fine-tuning multiple signaling pathways, including NF-κB, via substrate cleavage. Research indicates that targeting MALT1 scaffolding activity is likely the preferred approach for effectively suppressing NF-κB activity while minimizing impact on the immune system. Using our Smart AllosteryTM platform, we have developed potentially the first-in-class MALT1 scaffolding inhibitors targeting the CBM signalosome as a more directed approach to specifically block NF-κB signaling. Here, we disclose the distinctive mechanism of action of scaffolding inhibitors. Experiments: The mechanistic studies of MALT1 scaffolding inhibition were explored through comprehensive cellular assays and in vivo analyses. Engineered cell models were utilized to elucidate novel mechanisms of action. Results: We have developed a series of potent MALT1 scaffolding selective inhibitors with cellular IC50 values approximating 250nM for scaffolding activity, 80nM for NF-κB activity, and 590nM for AP-1 activity. Using TR-FRET, NanoBRET, and TurboID assays, we observed scaffolding inhibitors did not interrupt the interaction of MALT1-TRAF6. Rather, they induced a non-productive signalosome blocking NF-κB activity. Scaffolding inhibitors did not inhibit MALT1 protease activity; in fact, inhibition of MALT1 scaffolding activity modestly increased its protease activity, which is consistent with observations in MALT1 scaffolding mutant mice. This distinctive consequence of MALT1 scaffolding inhibition translated to no detrimental effects on T-cell activation or Treg differentiation, both of which are dependent on MALT1 protease activity and cause associated safety concerns with chronic MALT1 protease inhibitor treatment. The distinct and superior mode of action of MALT1 scaffolding inhibitors manifested in a more potent and broad anti-proliferative activity against a panel of B-NHL cell lines dependent on intrinsic BCR or NF-κB activity for growth. Conclusions: The potential first-in-class MALT1 scaffolding inhibitor offers what we believe is a best-in-class approach to targeting CBM-complex-driven NF-κB signaling. Scaffolding inhibitors effectively blocked both NF-κB and AP-1 signaling without impairing protease activity and avoided negative effects on T-cell function. Scaffolding inhibitors offer a promising therapeutic approach for NF-kB driven cancers. Citation Format: Yi Yao, Kevin Ling, Huili Xia, Huadong Sun, Yingzhi Bi, Jun Kuai, Joe Wahle, Wei Liu, Ken Carson, Gerry Harriman, Fang Wang. Targeting the CBM signalosome with a MALT1 scaffolding inhibitor for treatment of non-Hodgkin lymphomas [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-036.

Effect of AVM0703 treatment on the limitations of R-CHOP and survival in a model applicable to ABC subtype DLBCL.

e19060 Background: The current treatment regimen for DLBCL consisting of rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone (RCHOP), is challenged by molecular and prognostic characterization of DLBCL. Activated B-cell (ABC) lymphoma has a significantly worse prognosis (5 yr OS rate: 45% - 56%) than germinal center B-cell (GCB) lymphoma (78% - 80%) after RCHOP treatment. Around 40% of patients ultimately relapse and/or develop primary refractory disease, cardiotoxicity and long-term secondary malignancies. Efforts are being made to improve the RCHOP backbone by including dose intensification and by the addition of new agents to increase the efficacy and reduced toxicity of RCHOP (McKelvey EM Cancer 1976; Wilson WH Blood 2002; Molina TJ JCO 2014; Coiffier B Hematology 2016). AVM0703, an immunomodulatory drug, mobilizes bispecific γδ and inv-TCR double-positive novel T and NKT cells. In a previous study employing an aggressive, immune resistant A20 mouse lymphoma model (Bascuas T J Transl Med. 2016), neoadjuvant AVM0703 was additive/synergistic, when cyclophosphamide/fludarabine (CyFlu) was dosed 3 and 24 hr later, with no additional toxicities. In the present study we combined AVM0703 with RCHOP to evaluate the efficacy in A20 mice model. Methods: 10 week old Balb/c mice were injected with 2x106 murine A20 B-cells in the flank and were randomized (1:1 male: female) into 1) Placebo 2) 3 cycles RCHOP 3) 3 cycles AVM0703 (18mg/kg HED) 4) 3 cycles AVM0703+RCHOP, when tumor size reached ~100 mm3. Mice who achieved complete remission (CR) were re-challenged at 21-25 wks later. Pre and post re-challenge, presence of memory cells was analyzed using flow cytometer. All mice were monitored continuously as per the IACUC protocol. Results: Mice treated with the combination of AVM0703+RCHOP achieved superior CR compared to RCHOP alone, which was not curative in any mice. In first phase of the study, 3 out of 11 AVM0703 +RCHOP treated mice showed CR after only 1-cycle. In second phase, 2 of 12 mice survived in CR for >150 days after 3 cycles, whereas no RCHOP mice survived beyond 2 cycles. AVM0703+RCHOP treated mice in long-term CR, had memory cells (CD44hi-Cd62low) pre- and post re-challenge, and no A20 engraftment, suggesting strong immunological memory induced by AVM0703+RCHOP. Conclusions: AVM+RCHOP demonstrated anti-tumor activity in A20 lymphoma mice, with reduced toxicity, compared to RCHOP. Larger preclinical studies are underway to confirm the activity of AVM0703+RCHOP against RCHOP resistant ABC DLBCL, and the potential to reduce total RCHOP exposure to only 3 cycles while maintaining or improving efficacy. [Table: see text]

Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-Cell Lymphoma.

Pathomechanisms that activate oncogenic B-cell receptor (BCR) signaling in diffuse large B-cell lymphoma (DLBCL) are largely unknown. Kelch-like family member 6 (KLHL6) encoding a substrate-adapter for Cullin-3-RING E3 ubiquitin ligase with poorly established targets is recurrently mutated in DLBCL. By applying high-throughput protein interactome screens and functional characterization, we discovered that KLHL6 regulates BCR by targeting its signaling subunits CD79A and CD79B. Loss of physiologic KLHL6 expression pattern was frequent among the MCD/C5-like activated B-cell DLBCLs and was associated with higher CD79B levels and dismal outcome. Mutations in the bric-a-brac tramtrack broad domain of KLHL6 disrupted its localization and heterodimerization and increased surface BCR levels and signaling, whereas Kelch domain mutants had the opposite effect. Malfunctions of KLHL6 mutants extended beyond proximal BCR signaling with distinct phenotypes from KLHL6 silencing. Collectively, our findings uncover how recurrent mutations in KLHL6 alter BCR signaling and induce actionable phenotypic characteristics in DLBCL. Significance: Oncogenic BCR signaling sustains DLBCL cells. We discovered that Cullin-3-RING E3 ubiquitin ligase substrate-adapter KLHL6 targets BCR heterodimer (CD79A/CD79B) for ubiquitin-mediated degradation. Recurrent somatic mutations in the KLHL6 gene cause corrupt BCR signaling by disrupting surface BCR homeostasis. Loss of KLHL6 expression and mutant-induced phenotypes associate with targetable disease characteristics in B-cell lymphoma. See related commentary by Leveille et al. See related commentary by Corcoran et al.

The Oncogenic Lipid Sphingosine-1-Phosphate Impedes the Phagocytosis of Tumor Cells by M1 Macrophages in Diffuse Large B Cell Lymphoma.

A total of 30-40% of diffuse large B cell lymphoma (DLBCL) patients will either not respond to the standard therapy or their disease will recur. The first-line treatment for DLBCL is rituximab and combination chemotherapy. This treatment involves the chemotherapy-induced recruitment of tumor-associated macrophages that recognize and kill rituximab-opsonized DLBCL cells. However, we lack insights into the factors responsible for the recruitment and functionality of macrophages in DLBCL tumors. We have studied the effects of the immunomodulatory lipid sphingosine-1-phosphate (S1P) on macrophage activity in DLBCL, both in vitro and in animal models. We show that tumor-derived S1P mediates the chemoattraction of both monocytes and macrophages in vitro and in animal models, an effect that is dependent upon the S1P receptor S1PR1. However, S1P inhibited M1 macrophage-mediated phagocytosis of DLBCL tumor cells opsonized with the CD20 monoclonal antibodies rituximab and ofatumumab, an effect that could be reversed by an S1PR1 inhibitor. Our data show that S1P signaling can modulate macrophage recruitment and tumor cell killing by anti-CD20 monoclonal antibodies in DLBCL. The administration of S1PR1 inhibitors could enhance the phagocytosis of tumor cells and improve outcomes for patients.

Abstract C139: The IRAK4 inhibitor emavusertib (CA-4948) synergizes with second generation BTK inhibitors acalabrutinib and zanubrutinib in MYD88-L265P mutated lymphoma cell lines

Abstract Background. IRAK4 is a protein kinase downstream the Toll-like receptor (TLR), activated through the adaptor protein MYD88, crucial for the signaling cascade that leads to NF-κB-mediated cytokine and survival factor expression. Somatic mutations in the gene coding for the adaptor protein MYD88 are common in lymphoid tumors. In particular, the MYD88-L265P mutations, known to sustain NF-κB activation, is specifically observed in a fraction of the activated B cell (ABC) diffuse large B-cell lymphomas (DLBCL), in the vast majority of lymphoplasmacytic lymphoma (LPL), and in rare marginal zone lymphomas (MZLs). So far, no success has been achieved to directly inhibit MYD88. Emavusertib (CA-4948) is an IRAK4 inhibitor, which has shown preclinical activity in ABC DLBCL and MZL models, especially in cases bearing the MYD88-L265P. We and others have shown synergism when combining emavusertib with the first generation BTK inhibitor ibrutinib. Clinical trials are on-going exploring emavusertib as single agent or in combination in patients with hematological cancers or solid tumors. Since ibrutinib is associated with possible toxicities that can outbalance its clinical efficacy, here, we have tested the combination of emavusertib with acalabrutinib and zanubrutinib, two 2nd generation BTK inhibitors that have shown improved safety profile in clinical trials. Methods. Cell lines were exposed to five increasing concentrations of each compound as single agent or in combination for 72 h, followed by MTT assay. The beneficial effect of the combinations compared to the single agents was considered both as synergism according to the Chou-Talalay combination index and as potency and efficacy according to the MuSyC algorithm. Results. Cell lines bearing the MYD88-L265P mutation and derived from ABC DLBCL (OCI-Ly-10, TMD8, HBL1) or MZL/LPL (Karpas-1718) were exposed to emavusertib, acalabrutinib or zanubrutinib as single agent or in combination. Synergism, defined as a median Chou-Talalay index < 0.9, was observed for both the combination of emavusertib plus acalabrutinib and for the combination of emavusertib plus zanubrutinib in all four cell lines. Based on the MuSyC algorithm, the synergistic activity of emavusertib was mainly due to an improved efficacy (i.e., maximal effect) rather than improved potency (i.e., minimal active dose) of the BTK inhibitors. Conclusions. In MYD88-L265P mutated lymphoma cell lines, synergism is observed combining the IRAK4 inhibitor emavusertib with the 2nd generation BTK inhibitors acalabrutinib and zanubrutinib. Our data sustain further clinical exploration of the combination of IRAK4 and BTK inhibitors for lymphoma patients. Citation Format: Francesca Guidetti, Alberto J. Arribas, Eleonora Cannas, Emanuele Zucca, Anastasios Stathis, Reinhard von Roemeling, Francesco Bertoni. The IRAK4 inhibitor emavusertib (CA-4948) synergizes with second generation BTK inhibitors acalabrutinib and zanubrutinib in MYD88-L265P mutated lymphoma cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C139.

Final Results of a Phase I/II Trial of Carfilzomib + R-CHOP for Frontline Treatment of Patients with Non-Germinal Center Diffuse Large B-Cell Lymphoma (DLBCL)

INTRODUCTION: Recent data from the REMoDL-B trial show that addition of the proteasome inhibitor bortezomib to R-CHOP improves progression free survival (PFS) of patients (Pts) with activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL) compared to R-CHOP, but peripheral neuropathy limited dose intensity. Relative to bortezomib, carfilzomib (K) displays comparable clinical activity in plasma cell neoplasms but does not cause neurotoxicity. K has preferential clinical activity in relapsed non-germinal center (non-GC) DLBCL treated with chemotherapy (Torka, et al. Blood Adv. 2023). To explore the safety and efficacy of K in combination with R-CHOP (KR-CHOP), we performed a phase I trial to identify the recommended phase 2 dose (RP2D) in all DLBCL Pts followed by a phase II extension exclusively in non-GC DLBCL. METHODS: Between 2015-2020, Pts with untreated de novo or transformed DLBCL, adequate organ function and ECOG PS ≤ 3 were enrolled at 3 US academic medical centers. As previously reported [Hill, et al, Blood (2018), 132 (Supl.1):1692], during phase I dose escalation, the RP2D of K was 20 mg/m 2 on days (D) 1 & 2 of cycle 1 and 56 mg/m 2 on D1 & 2 of cycles 2-6. Rituximab was given on D2 and CHOP was given on D3. All Pts received pegfilgrastim and zoster prophylaxis. To assess the cardiac safety of combining K with anthracycline, we compared echocardiogram data at baseline and conclusion of therapy from study Pts and control NHL Pts treated with CHOP. To gauge for differential clinical activity of KR-CHOP vs R-CHOP, we compared PFS and overall survival (OS) rates for study Pts with non-GC DLBCL vs. Pts with non-GC DLBCL receiving standard of-care (SOC) R-CHOP at the Cleveland Clinic during the same period. The outcomes were also compared between propensity score (PS)-matched pairs, to account for differences in baseline demographics and clinical risk. RESULTS: 48 Pts were enrolled, and 47 treated; 18 in dose levels < RP2D and 29 at RP2D.Baseline demographics and treatment outcomes are shown in the Table. All-grade (G) hematologic adverse events (AEs) were: anemia, 35% (22% ≤ G2, 13% G3); thrombocytopenia, 24% (15% ≤ G2, 2% G3, 7% G4); febrile neutropenia, 9% (all G3/G4). Non-hematologic AEs included: nausea, 49% (47% ≤ G2, 2% G3); vomiting, 23% (21% ≤ G2, 2% G3), peripheral sensory neuropathy, 27% (all ≤ G2); dyspnea, 24% (all ≤G2). There was one case of G3 congestive heart failure. Echocardiograms from 42 study Pts who completed 6 cycles of KR-CHOP showed no statistically significant difference in change in ejection fraction from baseline compared to 39 control Pts [95% vs. 94%, respectively (P = 0.59)]. The overall response rate was 89% (70% CR, 19% PR). At a median follow-up of 31 months, 3-year Kaplan-Meier estimates of PFS and OS for the study population were 79% and 87%, respectively. There were 8 deaths in the study: 4 due to lymphoma; 2 due to infection; 2 due to other causes. Comparing outcomes of the 36 study Pts with non-GC DLBCL to 172 control Pts receiving R-CHOP as SOC, more control Pts had stage IV disease (54% vs 34%, p=0.03) and older age (median 67 vs 66 years old, p=0.03), but less frequently >1 extranodal site of disease (23% vs. 42%, P = 0.02). PS matching using logistic regression identified 25 case-control pairs of Pts with non-GC DLBCL according to gender, age, and other International Prognostic Index (IPI) elements (see Table). In analysis of PS-matched pairs of Pts with non-GC DLBCL, Pts treated with KR-CHOP had better 3-year PFS and OS relative to Pts treated with R-CHOP as SOC [96% vs. 50% (p=0.002) (see Figure) and 67% vs. 96% (p = 0.02), respectively]. In Cox univariate analysis, treatment with R-CHOP vs. KR-CHOP was associated with an increased risk of PFS and OS with hazard ratios (HR) of 6.15 [95% confidence interval (CI) 1.71 - 22.05, P = 0.002] and 3.19 [(95% CI, 1.01 -10.11), P = 0.02], respectively. CONCLUSION: KR-CHOP can be delivered safely, without excess cardiac toxicity or peripheral neuropathy. The superior PFS and OS rates for Pts with non-GC DLBCL vs. controls receiving R-CHOP are encouraging but should be interpreted with caution due to inherent selection bias in clinical trial populations. These data suggest that, like bortezomib, carfilzomib (K) may have preferential clinical activity in ABC DLBCL. Future randomized trials are justified using advanced molecular profiling to further investigate which Pts with DLBCL may benefit from addition of a proteasome inhibitor as part of frontline treatment.

Genomic Stability, DNA Repair and the SKP1-CUL1-F-Box Complex in HIV Associated Diffuse Large B-Cell Lymphoma

Introduction: Previously we demonstrated that HIV associated diffuse large B-cell lymphomas (DLBCL) of Germinal Center B-cell subtype (GCB) are more proliferative, with enhanced genomic stability and increased expression of DNA repair genes, particularly Fanconi anemia (FA) and homologous recombination (HR) genes compared to their HIV negative counterparts (Maguire et al, 2019). It is unknown if this signature is unique to the GCB subtype or if it is also observed in Activated B-cell (ABC) and Unclassified (UNC) DLBCL. Herein we validate our HIV associated GCB DLBCL findings in a larger independent cohort and report for the first time the aberrational profile of molecularly profiled HIV associated ABC DLBCL compared to their HIV negative counterparts respectively. Methods: An independent cohort of 353 DLBCL FFPE cases was assembled, including 125 HIV-DLBCL samples. All samples underwent hematopathological review to confirm diagnosis and determine tumor content. Samples with <60% tumor content were macro-dissected before they were RNA/DNA extracted. Cell of origin (COO) profiles were assessed using the Lymph3Cx molecular assay which can distinguish primary mediastinal B-cell lymphoma (PMBL) like from GCB, ABC and UNC DLBCL (Mottok et al 2018). Copy number (CN) analyses were performed on samples with sufficient DNA (41 HIV neg, 32 HIV pos) using the OncoScan FFPE assay kit and the resulting data analyzed using Chromosome Analysis Suite (ChAS) v4.1 and the multisample viewer module of Reproductive Health Research Analysis Software (RHAS) v1.1. Additional digital gene expression profiling was performed using the Pan-Cancer panel (NanoString, Seattle, WA) customized to include 9 BCL2 family genes for a total of 739 genes and analyzed using the NanoString nSolver Advanced Analysis module. Results: GCB, ABC, UNC and PMBL-like proportions were 50%, 30%, 14% and 6% in HIV unassociated (HIV neg) and 50%, 23%, 25% and 2% in HIV pos tumors respectively, confirming both cohorts were GCB-enriched by molecular profiling. PMBL-like cases were excluded from further analyses. OncoScan CN analyses showed that HIV pos DLBCL were significantly less aberrant than HIV neg DLBCL (median CN per sample 0.56 vs 1.01, p<0.001). HIV pos GCB tumors were significantly less aberrant than both HIV neg GCB tumors (median CN per sample 0.50 vs 1.11, p<0.001), and HIV pos ABC tumors (median CN per sample 0.50 vs 0.81, p=0.009). HIV pos ABC tumors were also less aberrant than HIV neg ABC tumors, however, the difference was not significant overall (median CN per sample 0.81 vs 1.03, p=0.162) or for gain/amplifications (median CN per sample 0.38 vs 0.42, p=0.814), but was borderline significant for loss/deletions (median CN per sample 0.31 vs 0.47, p=0.06). GEP analyses identified Cell Cycle and DNA repair as the top two upregulated pathways in HIV pos GCB tumors compared to HIV neg GCB tumors. In contrast, Cell Cycle and DNA repair were downregulated in HIV pos ABC tumors compared to HIV neg ABC tumors. However, one gene ( RBX1) was identified as significantly upregulated in both pathways in HIV pos GCB (adjusted p-value 0.0215) and HIV pos ABC (adjusted p-value 0.0046) tumors compared to their HIV neg counterparts. A second gene ( SKP1) was also significantly upregulated in HIV pos GCB (adjusted p-value 3.94E-5) and HIV pos ABC (adjusted p-value 0.0031) tumors. Together RBX1 and SKP1 encode the two principal functional subunits of the SKP1, CUL1, F-box protein (SCF) complex which is part of the ubiquitin proteasome system (UPS) and has been reported as essential for maintaining genome and chromosome stability (Thompson et al 2021). Conclusions: These results demonstrate that HIV pos DLBCL tumors are less aberrant than their HIV neg counterparts irrespective of COO subtype. However, the degree of genomic stability differs by COO, with HIV pos GCB tumors being more genomically stable than HIV pos ABC tumors. Although both have enhanced expression of UPS related SCF genes ( RBX1, SKP1), only HIV pos GCB tumors have enhanced expression of key DNA repair genes as well including Fanconi Anemia ( BRCA2/FANCD1, FANCL) and mismatch repair ( MSH2, MSH6) genes (adjusted p-values of ≤ 0.0263). Given the reported role of SCF in genomic stability maintenance, the HIV pos COO CN differences could be due to the contributions of both DNA repair and SCF mechanisms in HIV pos GCB and mainly SCF mechanisms in HIV pos ABC tumors.

Combination Epigenetic Therapy Using YF2, a First-in-Class Histone Acetyltransferase Activator, Restores Immunogenicity in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. Molecular studies have identified epigenetic dysregulation as a key driver in germinal center (GC)-derived DLBCL. Mutations in CREBBP/p300 and EZH2 often co-occur and lead to transcriptional repression and decreased immunogenicity as demonstrated by reduced major histocompatibility complex (MHC) expression. Our lab previously reported that YF2, a first-in-class histone acetyltransferase activator discovered at Columbia University, induces cytotoxicity in both cell and murine models of DLBCL. Other epigenetic therapies, such as tazemetostat and belinostat, are approved for use in follicular and T-cell lymphoma, respectively, but have limited single agent activity in DLBCL. We hypothesize that treatment with YF2 in combination with tazemetostat or belinostat increases transcriptional activity and restores tumor immunogenicity in CBP/p300- and EZH2-mutated DLBCL. We utilized several DLBCL cell lines to assess YF2 combination therapy: OCI-Ly7 (CBP/p300 WT, EZH2 WT), SU-DHL-4 (CBP/p300 WT, EZH2 GOF), A20 (CBP/p300 -/-, EZH2 WT), and SU-DHL-6 (CBP/p300 -/-, EZH2 GOF). Cells were treated on day 0 with single agents or with YF2 in combination with either tazemetostat (YF2 + taz) or belinostat (YF2 + bel), retreated on day 3, and collected on day 6 for flow cytometry (n = 4) and western blot (n = 2) analyses. MHC class I/II and β₂ microglobulin (B2M) expression were measured as markers of immunogenicity. To preliminarily assess YF2 combination therapy in vivo, immunocompetent BALB/c mice were engrafted with syngeneic A20 cells, immunized, and segregated (n = 2-4) into treatment groups: control (saline), tazemetostat (300 mg/kg twice daily x 28 days), belinostat (40 mg/kg once daily x 7 days), YF2 (50 mg/kg once daily x 28 days), YF2 + taz, and YF2 + bel. Blood and spleen samples were collected on day 29 to measure T- and B-cell activity via flow cytometry. All fold changes are expressed relative to the corresponding control. Combination therapy generally increased MHC-I, -II, and B2M expression across all cell lines as measured via flow cytometry. In double-mutated SU-DHL-6 cells, MHC-I and -II expression increased 2.0- ( P < 0.01) and 3.1-fold ( P = 0.04), respectively, following YF2 + taz treatment, and increased 1.3- ( P = 0.06) and 4.5-fold ( P < 0.01) after YF2 + bel ( Figure 1). Expression of B2M increased 2.3-fold ( P = 0.01) with YF2 + taz and 1.6-fold ( P = 0.07) with YF2 + bel. Interestingly, YF2 combination therapy was similarly effective in non-mutated OCI-Ly7 cells: MHC-I, -II, and B2M expression increased 1.9- ( P < 0.01), 3.0- ( P < 0.01), and 2.4-fold ( P < 0.01), respectively, following YF2 + taz treatment, and increased 1.5- ( P = 0.01), 3.3- ( P < 0.01), and 2.4-fold ( P < 0.01) with YF2 + bel. Similar expression trends were observed via western blots, but to a lesser degree; MHC-I, -II, and B2M protein increased 1.3-, 1.4-, 1.6-fold with YF2 + taz, and increased 1.1-, 1.6-, and 1.2-fold following YF2 + bel treatment in SU-DHL-6 cells. The combination therapies were tolerated by the mice and decreased tumor size. Splenic MHC-I expression increased 1.2-fold with YF2 + taz and 1.4-fold with YF2 + bel, whereas MHC-II increased 1.3-fold for both combination therapies ( Figure 2). Similarly, circulating B cells displayed increased MHC-I and -II expression with combination therapy. Treatment with YF2 + taz increased the number of circulating CD4 + T cells 1.5-fold and the number of CD8 + T cells 3.5-fold, while YF2 + bel increased the number of central memory and effector memory T cells by 1.6- and 1.9-fold, respectively. To understand the effect of combination therapies on B cell development in the GC, splenic B cells were evaluated and exhibited a 1.9-fold increase of cells in the light zone following YF2 + taz treatment, and a 1.2-fold increase after YF2 + bel exposure. The number of splenic plasma cells increased 1.4- and 1.5-fold following YF2 + taz and YF2 + bel treatments, respectively ( Figure 2). These findings suggest that combination epigenetic therapy may restore homeostatic B cell function and promote B cell cycling through the GC. In summary, these data propose YF2 combination epigenetic therapy as a means to increase immunogenicity in CBP/p300- and EZH2-mutated DLBCL. While our preliminary mouse experiment provided promising initial results, an expanded study is underway to statistically confirm the exciting trends that we observed.

Leukemic presentation and progressive genomic alterations of MCD/C5 diffuse large B-cell lymphoma (DLBCL).

Diffuse large B-cell lymphoma (DLBCL) is a heterogenous group of lymphoid malignancies. Based on gene expression profiling, it has been subdivided into germinal center (GC)-derived and activated B-cell (ABC) types. Advances in molecular methodologies have further refined the subclassification of DLBCL, based on recurrent genetic abnormalities. Here, we describe a distinct case of DLBCL that presented in leukemic form. DNA sequencing targeting 275 genes revealed pathogenically relevant mutations of CD79B, MyD88, TP53, TBL1XR1, and PIM1 genes, indicating that this lymphoma would be best classified as MCD/C5 DLBCL, an ABC subtype. Despite an initial good clinical response to BTK inhibitor ibrutinib, anti-CD20 antibody rituxan, alkylating agent bendamustine, and hematopoietic stem-cell transplant, the lymphoma relapsed, accompanied by morphologic and molecular evidence of disease progression. Specifically, the recurrent tumor developed loss of TP53 heterozygosity (LOH) and additional chromosomal changes central to ABC DLBCL pathogenesis, such as PRDM1 loss. Acquired resistance to ibrutinib and rituxan was indicated by the emergence of BTK and FOXO1 mutations, respectively, as well as apparent activation of alternative cell-activation pathways, through copy-number alterations (CNAs), detected by high-resolution chromosomal microarrays. In vitro, studies of relapsed lymphoma cells confirmed resistance to standard BTK inhibitors but sensitivity to vecabrutinib, a noncovalent inhibitor active against both wild-type as well as mutated BTK. In summary, we provide in-depth molecular characterization of a de novo leukemic DLBCL and discuss mechanisms that may have contributed to the lymphoma establishment, progression, and development of drug resistance.

Phase 1 Study of Acalabrutinib Plus Danvatirsen (AZD9150) in Relapsed/Refractory Diffuse Large B-Cell Lymphoma Including Circulating Tumor DNA Biomarker Assessment.

Novel targeted and immunotherapies have improved outcomes in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), but toxicities limit widespread use. The selective Bruton tyrosine kinase (BTK) inhibitor acalabrutinib has activity in patients with R/R DLBCL, but durable responses are uncommon. STAT3 inhibition has demonstrated clinical activity in DLBCL. Final results of the Phase 1 study of acalabrutinib plus STAT3 inhibitor (danvatirsen; AZD9150) in patients with R/R DLBCL are reported. Danvatirsen 200 mg intravenous infusion (Days 1, 3, 5 [Cycle 1]; weekly infusions starting Day 8, Cycle 1) was administered in combination with oral acalabrutinib 100 mg twice daily until progressive disease (PD) or unacceptable toxicity. Primary endpoints were safety and tolerability. Secondary endpoints included efficacy, pharmacokinetics, and immunogenicity. Seventeen patients received combination treatment. One dose-limiting toxicity (Grade 3 liver transaminase) occurred in one patient. The most common reason for treatment discontinuation was PD (65%). In evaluable patients (n=17), objective response rate was 24%; median duration of response was 1.9 months. All responders with available DLBCL cell-of-origin data were either activated B-cell or nongerminal center B-cell like subtype. Genetic subtype did not correlate with response. Baseline and longitudinal plasma cell-free DNA (cfDNA) concentrations were mostly higher in nonresponding patients. cfDNA changes were generally concordant with imaging. Pretreatment circulating B-cell levels were higher in responders versus nonresponders. Targeting both STAT3 and BTK in combination is safe and tolerable but efficacy is limited in R/R DLBCL. Results support evaluation of circulating tumor DNA as a biomarker for clinical response.

Abstract 3689: Uncovering the metabolic vulnerabilities in activated B cell diffuse large B cell lymphoma

Abstract DLBCL is the most common B cell malignancy in adults accounting for 30-40% of non-Hodgkin lymphoma. Only 60% of these cases are curable, with activated B cell DLBCL (ABC-DLBCL) having inferior outcomes than the germinal center (GCB) DLBCL. The use of ibrutinib to treat non-responsive/relapsed ABC-DLBCL cases has significantly improved the treatment of this disease. However, in more than 40% of ABC-DLBCL cases, the disease progresses or develops resistance to ibrutinib. Thus, there is an urgent unmet need to understand the molecular basis of resistance mechanisms and identify novel therapeutic regimens to treat IR DLBCL. Utilizing high throughput metabolomics, we identified cysteine-methionine metabolism and alanine, aspartate, and glutamate metabolism to be deregulated in IR cells. We further identified interleukin four induced 1 (IL4I1), an L-amino acid oxidase that partakes in cysteine and methionine metabolism by contributing to the methionine salvage pathway. The amino acid cysteine contributes to cancer metabolic remodeling by a), in its free form, in redox control, as a component of the antioxidant glutathione or its involvement in protein s-cysteinylation, b), as a substrate for the production of hydrogen sulfide (H2S), which feeds the mitochondrial electron transfer chain and mediates persulphidation of ATPase and glycolytic enzymes, thereby stimulating cellular bioenergetics; and, c), as a carbon source for epigenetic regulation, biomass production, and energy production. Consistent with this, we show that loss of IL4I1 expression induced aberrant cysteine and methionine metabolism accompanied by metabolic reprogramming to OXPHOS, which collectively enabled increased drug tolerance of DLBCL. This adaptive metabolic mechanism exhibited increased cysteine and cystathionine intracellular levels in IR-DLBCL lacking IL4I1 expression. As cysteine and glutathione are scavengers of free radicals (mainly reactive oxygen species (ROS)), they can nullify the effects of the majority of oxidative or alkylating drugs used in cancer therapy, affording a vital resistance mechanism and disruption of cysteine homeostasis appears to be promising strategy for induction of cancer cell death and provide therapeutic approaches to treat Ibrutinib-resistant-ABC- DLBCL for improved patient care. Citation Format: Satishkumar Singh, Fouad Choueiry, Anuvrat Sircar, Narendranath Epperla, Jiangjiang Zhu, Lalit Sehgal. Uncovering the metabolic vulnerabilities in activated B cell diffuse large B cell 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 3689.

CDK9 inhibition induces epigenetic reprogramming revealing strategies to circumvent resistance in lymphoma

Diffuse large B-cell lymphoma (DLBCL) exhibits significant genetic heterogeneity which contributes to drug resistance, necessitating development of novel therapeutic approaches. Pharmacological inhibitors of cyclin-dependent kinases (CDK) demonstrated pre-clinical activity in DLBCL, however many stalled in clinical development. Here we show that AZD4573, a selective inhibitor of CDK9, restricted growth of DLBCL cells. CDK9 inhibition (CDK9i) resulted in rapid changes in the transcriptome and proteome, with downmodulation of multiple oncoproteins (eg, MYC, Mcl-1, JunB, PIM3) and deregulation of phosphoinotiside-3 kinase (PI3K) and senescence pathways. Following initial transcriptional repression due to RNAPII pausing, we observed transcriptional recovery of several oncogenes, including MYC and PIM3. ATAC-Seq and ChIP-Seq experiments revealed that CDK9i induced epigenetic remodeling with bi-directional changes in chromatin accessibility, suppressed promoter activation and led to sustained reprograming of the super-enhancer landscape. A CRISPR library screen suggested that SE-associated genes in the Mediator complex, as well as AKT1, confer resistance to CDK9i. Consistent with this, sgRNA-mediated knockout of MED12 sensitized cells to CDK9i. Informed by our mechanistic findings, we combined AZD4573 with either PIM kinase or PI3K inhibitors. Both combinations decreased proliferation and induced apoptosis in DLBCL and primary lymphoma cells in vitro as well as resulted in delayed tumor progression and extended survival of mice xenografted with DLBCL in vivo. Thus, CDK9i induces reprogramming of the epigenetic landscape, and super-enhancer driven recovery of select oncogenes may contribute to resistance to CDK9i. PIM and PI3K represent potential targets to circumvent resistance to CDK9i in the heterogeneous landscape of DLBCL.

Ars2-containing bispecific, Fab- and IgG1-format BAR-bodies to target DLBCL cells.

Despite recent advances in the therapy of diffuse large B-cell lymphoma, not otherwise specified (DLBCL), around 30% of patients develop refractory disease or relapse after first-line treatment. Recently, Ars2 was reported as the auto-antigenic target of the B-cell receptor (BCR) in approximately 25% of activated B-cell DLBCL cases. Ars2 could be used to specifically target B cells expressing Ars2-reactive BCRs. However, the optimal therapeutic format to integrate Ars2 into has yet to be determined. To mimic therapeutic antibody formats, Ars2-containing bispecific and IgG1-like constructs (BCR antigens for reverse [BAR]-bodies) were developed. Two bispecific BAR-bodies connecting single-chain antibodies against CD16 or CD3 to the BCR-binding epitope of Ars2 were constructed. Both constructs showed strong binding to U2932 cells and induced effector cell-dependent and selective cytotoxicity against U2932 cells of up to 44% at concentrations of 20μg/ml. Additionally, IgG1-format Ars2 BAR-bodies were constructed by replacing the variable heavy- and light-chain regions of a full-length antibody with the Ars2 epitope. IgG1-format Ars2 BAR-bodies also bound selectively to U2932 and OCI-Ly3 cells and induced selective cytotoxicity of up to 60% at 10μg/ml. In conclusion, Ars2-containing bispecific and IgG1-format BAR-bodies both are new therapeutic formats to target DLBCL cells.

Phase 1 Study of Venetoclax, Ibrutinib, Prednisone, Obinutuzumab, and Lenalidomide in Combination with Polatuzumab (ViPOR-P) in Relapsed/Refractory B-Cell Lymphoma: Preliminary Analysis of Safety and Efficacy

Background: Diffuse large B-cell lymphoma (DLBCL) is often curable with frontline chemotherapy; however, pts with relapsed/refractory (R/R) disease and those who fail CAR-T therapy have poor overall outcome. Oral small molecule targeted agents can disrupt key survival pathways in DLBCL, but fail to induce deep or durable responses as monotherapy. We have previously shown that ViPOR is safe and active in DLBCL, with preferential activity in non-GCB DLBCL and HGBCL-DH (Melani et al. Blood. 2020). Synergistic activity has also been demonstrated with MMAE, the chemotherapy payload of Polatuzumab (Pola), with ViPOR in pre-clinical DLBCL cell lines (unpublished data). We hypothesized that additional genotoxic stress with Pola will further leverage efficacy and time-limited, cyclic dosing will limit the hematologic toxicity of the regimen. Methods: R/R aggressive B-cell lymphoma pts with adequate organ function were eligible. In phase 1, a 3+3 design was used to determine the maximum tolerated dose (MTD) of 3 dose-levels (DLs) of Pola IV D2 and venetoclax (Ven) PO D2-14 (DL1: 1.4mg/kg + 600mg, DL2: 1.8mg/kg + 600mg, and DL3: 1.8mg/kg + 800mg) in combination with fixed-dose ibrutinib 560mg PO D1-14, prednisone 100mg PO D1-7, obinutuzumab 1000mg IV D1-2, and lenalidomide 15mg PO D1-14. An expansion cohort was included at the MTD for further analysis of safety and efficacy. ViPOR-P q21d x 6 cycles (C) was given without maintenance or planned consolidation. All pts received TLS, PCP, HSV, and G-CSF prophylaxis (ppx) with VTE ppx per investigator discretion. Baseline CT, PET, BM, and tumor biopsy were performed with CT scans after C2, 4, and 6 and PET after C6 or at time of suspected complete response (CR). Surveillance CT was performed q3m for 1y, q4m x 1y, q6m x 1y, then annually x 2y. Results: 15 pts were enrolled and treated in the dose-escalation cohort; 3 at DL1, 6 at DL2, and 6 at DL3. Lymphoma subtypes included DLBCL NOS (60%) and HGBCL-DH (40%). In 9 DLBCL NOS pts, 11% were GCB and 89% non-GCB subtype by IHC. Three (20%) pts had transformed disease. Median age was 51y (range 39-83), with stage III/IV disease in 80%, elevated LDH in 80%, and >2 extranodal sites of disease in 67%. Nine (60%) pts had high-intermediate/high-risk IPI. Median prior therapies were 2 (range 1-6), with prior BTKi in 27%, Pola in 7%, and CAR-T in 20%. Ten (67%) pts were primary refractory (i.e., <CR to frontline tx) and 8 (53%) of pts were refractory (i.e., <PR) to last tx. One dose-limiting toxicity (DLT) of prolonged G4 thrombocytopenia occurred at DL3, but no other DLTs were noted. Pola 1.8mg/kg and Ven 800mg were identified as the MTD and safe doses for use in the expansion cohort. Heme AEs were most common and included G3-4 (% cycles) thrombocytopenia (17%), anemia (7%), and neutropenia (5%). No cases of febrile neutropenia occurred across 58 cycles. The only non-heme G3-4 AE occurring in >10% pts was hypokalemia (29%), with all pts experiencing any grade hypokalemia requiring replacement. Other common non-heme AEs (% pts) included diarrhea (71%), increased LFTs (64%), nausea (50%), hypomagnesemia (43%), and rash (43%). VTE occurred in 7% with no a.fib or major bleeding observed. No events of TLS or treatment-related mortality occurred. Dose reductions and delays occurred in 3% and 5% of cycles, respectively. Of 12 pts off-tx, 7 completed all 6C, 3 experienced disease progression, and 1 each came off-tx per investigator discretion or unacceptable AE. Of 15 enrolled pts, 14 are evaluable for response with an overall response rate (ORR) and CR rate of 64% (9/14) and 36% (5/14), respectively (Fig 1A). With a median potential f/u of 7.4m, median TTR in responding pts was 1.35m, with 8 (89%) of 9 total responses ongoing (Fig 1B). One HGBCL-DH pt relapsed by imaging at 10.6m, 2 PR pts received consolidative XRT with allo-HSCT, and 2 PR pts remain on-tx. PFS and OS at 6m was 69.8% and 75%, respectively. Conclusion: ViPOR-P is safe without significant additional toxicity compared to ViPOR, and Pola 1.8mg/kg and Ven 800mg were taken forward in the ongoing expansion cohort. Most common G3-4 AEs were hematologic with no febrile neutropenia observed with the use of G-CSF ppx. Fixed-duration ViPOR-P x 6C without maintenance led to CR in 5 (42%) of 12 off-tx DLBCL pts with 80% CRs ongoing from 3.7-9.3m. A randomized phase 2 trial is planned to further elucidate the safety and efficacy of Pola in combination with ViPOR across R/R DLBCL and within pre-defined molecular and genetic subtypes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel Autochthonous Mouse Models of Serve As Preclinical Tools to Investigate Pathogenesis and Treatment of MCD/C5 DLBCL

Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Both, biologically and clinically, DLBCL represents a highly heterogeneous disease. Based on gene expression profiling DLBCL is subdivided into germinal center B cell (GCB)-like and non-GCB- or activated B cell (ABC)-like DLBCL, a classifier which distinguishes DLBCL sub-entities displaying distinct biological features and clinical response to frontline anthracycline-based chemo-immune therapy. Plasmablasts have long been regarded as the cell of origin (COO) of ABC DLBCL, however, recent evidence points towards aberrant memory B cells (MB) as the true COO of these lymphomas. Several recent studies attempted to give structure to the genetic heterogeneity observed in DLBCL by clustering cases based on their mutational profiles. Clusters MCD by Schmitz et al. and C5 by Chapuy et al. are amongst the clusters enriched for ABC-DLBCL cases with particularly inferior prognosis. Both clusters are defined by mutations in MYD88 and CD79B as well as high levels of BCL2 expression and recurring mutations in transcriptional regulators of the plasmacytic differentiation process (PRDM1, SPIB). We previously reported an autochthonous mouse model of DLBCL mimicking hallmark mutations of MCD/C5 DLBCL: Combination of Myd88 p.L252P (the murine orthologue of human MYD88 L265P) and overexpression of BCL2 leads to the generation of ABC-DLBCL-like lymphomas in the Myd88p.L252P/wt;R26LSL.BCL2/wt;Cd19Cre/wt (MBC) mouse line. MBC Lymphoma cells show a plasmablastic immunophenotype with expression of CD138 and lack of expression of B220/CD19. In an attempt to refine our genetically-engineered mouse model, we incorporated other key alterations of MCD/C5 DLBCL in the MBC background: B cell specific loss of PRDM1, overexpression of SPIB and hallmark mutations in CD79B. Young Prdm1fl/fl;Myd88p.L252P/wt;R26LSL.BCL2/wt;Cd19Cre/wt (PPMBC) and Myd88p.L252P/wt;R26LSL.BCL2/LSL.Spib;Cd19Cre/wt (SMBC) compound animals display a strong reduction of the CD138+ population compared to control, accompanied by a decrease in serum immunoglobulins, in agreement with a block in plasma cell differentiation mediated by these additional genetic alterations. In contrast, cells with a marker profile of age-associated B cells (ABC, B220+/CD19+/CD21-/CD23-) as well as Memory B cells (MB, B220+/IgD-/Fas-/CD38+) accumulate markedly in all lymphoma lines, but particularly in PPMBC animals, compared to control. This is in line with recent studies hinting at ABC/ MB as the cells of origin of ABC-DLBCL. While MBC lymphomas are largely CD138+ and represent a plasmablastic/-cytic differentiation stage, PPMBC tumors express B-cell markers B220 and CD19 and lack expression of CD138. Additionally, PPMBC lymphomas frequently show a marker profile reminiscent of memory B cells, while CD21/CD23 - negativity suggests a relation to age-associated B cells, a population enlarged also in the pre-malignant setting. PPMBC tumors furthermore show an enrichment for B-cell receptor (BCR), NFkB and PI3K signaling gene signatures, when compared to MBC lesions. These signs of enhanced B cell receptor signaling translate into an increased sensitivity towards inhibition of Bruton's Tyrosine Kinase (BTK) by ibrutinib. The sensitivity of ibrutinib is further enhanced in lymphomas additionally expressing Cd79b p.Y195H, both in the Prdm1-deficient and the Prdm1-proficient model systems. We have established and characterized novel refined autochthonous mouse models of MCD/C5 DLBCL which serve as a preclinical tool to test novel therapeutic strategies in this sub-entity and pave the way to a deeper understanding of molecular mechanisms of pathogenesis, treatment response and resistance in highly aggressive DLBCL.

CFT7455, a Novel IKZF1/3 Degrader, Demonstrates Potent Anti-Tumor Activity in Models of Non-Hodgkin's Lymphoma As a Single Agent or in Combination with Clinically Approved Agents

Immunomodulatory drugs, such as pomalidomide and lenalidomide are approved for the treatment of multiple subtypes of non-Hodgkin's lymphoma (NHL). These drugs induce an interaction between Ikaros family zinc finger proteins 1 and 3 (IKZF1/3) and cereblon (CRBN), an E3 ligase, resulting in IKZF1/3 degradation. IKZF1/3 are master transcription factors that play a key role in B and T-cell differentiation and maintain levels of interferon regulatory factor 4 (IRF4), a critical regulator in NHL. Clinically, immunomodulatory drugs have shown activity as single agents and when used in combination with several classes of targeted therapies, such as rituximab, in both first line and relapsed/refractory NHL subtypes. We previously described the preclinical characterization of CFT7455 as a novel IKZF1/3 degrader with 800-1600-fold increased potency over pomalidomide in CRBN binding and cellular IKZF1 degradation assays. We demonstrated that this increased activity translated into dramatically greater efficacy compared to pomalidomide in cellular and in vivo preclinical models of multiple myeloma and NHL. Here we show that the increased catalytic activity and improved potency of CFT7455 also translates into potent anti-tumor activity as single agent or in combination with clinically approved agents in central nervous system (CNS) and subcutaneous models of NHL. In both the Raji and OCI-Ly10 CNS xenograft models, CFT7455 (100 µg/kg/day) led to a significant increase in survival in comparison to pomalidomide (P=0.002, P=0.0002). The combination of immunomodulatory drugs with CD20, BTK, or HDAC targeting agents shows promising clinical activity in the treatment of NHL. Combination studies were conducted, dosing CFT7455 in combination with rituximab, ibrutinib, or the HDAC inhibitor romidepsin in several models of NHL. In the mantle cell lymphoma (MCL) model, Mino, the combination of CFT7455 with rituximab demonstrated synergistic activity and complete tumor regression. In the diffuse large B-cell lymphoma (DLBCL) model, TMD8, the combination of ibrutinib and CFT7455 was synergistic and resulted in a significant increase in survival probability. In the CNS NHL model, OCI-Ly10, the combination of CFT7455 with rituximab or ibrutinib also led to a significant increase in survival probability highlighting the therapeutic potential of this combination. Synergistic activity was also observed with the combination of CFT7455 and romidepsin in two models of anaplastic large cell lymphoma (ALCL). In conclusion, CFT7455 is a potent, selective catalytic degrader of IKZF1/3, with single agent and combination antitumor activity in DLBCL, ALCL, and MCL models, supporting clinical investigation of CFT7455 for NHL.

Mivavotinib, a Syk Inhibitor, in Relapsed/Refractory (R/R) Non-GCB Diffuse Large B-Cell Lymphoma (DLBCL) with or without MYD88 and/or CD79 Mutations: A Phase 2 Study

Background: Spleen tyrosine kinase (Syk) is a key signaling protein that activates survival pathways in B-cell lymphomas. The non-germinal center B-cell like (non-GCB) or activated B-cell like (ABC) cell-of-origin subtype of DLBCL has greater reliance on the B-cell receptor pathway and Syk signaling for survival, compared with the GCB subtype. Multiple signaling pathways are activated by Syk in ABC DLBCL (NF-κB and phosphoinositide 3-kinase [PI3K]), whereas only the PI3K pathway is activated by Syk in GCB DLBCL. MYD88 and/or CD79b mutations are strongly enriched within ABC DLBCL and are associated with poor prognosis. Syk hyperactivation has been observed in ABC DLBCL cell lines harboring MYD88/CD79 mutations, with activation of NF-κB via Syk-mediated signaling through both the BCR and the toll-like-receptor (TLR)-MYD88 pathways. Mivavotinib is an orally dosed, potent, and selective Syk inhibitor, which showed potent single agent activity in preclinical studies in ABC and GCB DLBCL cell lines, with the strongest activity against those harboring MYD88 and/or CD79b mutations. In the clinical setting, heavily pre-treated all-comer R/R DLBCL patients receiving single agent mivavotinib at the recommended phase 2 dose (100 mg once daily [QD]) showed an overall response rate (ORR) of 33% (16% complete responses [CR]) with durable responses (median duration of response [DoR] not reached [95% CI: 7 months - NE]) and a favorable safety profile. Subgroups by cell-of-origin showed a substantially higher ORR in patients with the non-GCB subtype (53%; 95% CI: 27, 79) than GCB (22%; 95% CI: 0.11, 0.37), and a median DoR of 15.7 months (95% CI: 2.2, NE) in non-GCB responders. CX-659-401 (NCT05319028) is an ongoing study evaluating two dosing regimens of mivavotinib monotherapy in patients with R/R non-GCB DLBCL, in whom circulating tumor DNA-based next generation sequencing (NGS) will be used to determine MYD88/CD79b mutational status. The goals of this strategy are to confirm the activity of mivavotinib previously observed in non-GCB DLBCL, evaluate its activity in DLBCL according to mutational status of MyD88/CD79b, and optimize the dose and schedule in this biomarker-defined patient population. Methods: This multicenter, open-label, phase 2 study will enroll patients with histologically confirmed de novo or transformed DLBCL that is non-GCB by immunohistochemistry (IHC) per Hans algorithm or ABC by gene expression profiling, including primary mediastinal and primary extranodal DLBCL. Patients must have had at least 2 but no more than 5 prior lines of systemic anticancer therapy. Following standard first-line R-CHOP (or equivalent), patients must have received 2nd-line salvage therapy with or without autologous stem cell transplant (ASCT), and/or CAR-T therapy, unless ineligible for 2nd-line salvage, ASCT, or CAR-T. Additional key eligibility criteria include age >18 years, ECOG performance status 0-2, and FDG-PET-avid measurable disease per 2014 Lugano criteria. DLBCL patients with primary brain or secondary central nervous system involvement with active disease will be excluded. Patients will be randomized to 1 of 2 arms (n=25 planned per arm): (1) 100 mg QD; or (2) 120 mg QD for 2 weeks followed by 80 mg QD thereafter; in 28-day cycles until disease progression or unacceptable toxicity. The primary endpoints are ORR (by independent radiology review according to 2014 Lugano Criteria) and safety/tolerability. Secondary endpoints include DoR, progression-free survival (PFS), overall survival (OS), and CR rate. Exploratory endpoints include pharmacokinetic, biomarker assessments, and investigator-assessed ORR, DoR, PFS, and CR rate. Results of this study will help to define the optimal dose and provide confirmatory efficacy and safety data of mivavotinib in patients with non-GCB DLBCL, including MYD88/CD79b-mutated DLBCL to guide further expansion in these biomarker-defined cohorts.

Phase 1 Study of KT-413, a Targeted Protein Degrader of IRAK4 and IMiD Substrates, in Adult Patients with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

Background: Oncogenic mutations in myeloid differentiation primary response 88 (MYD88) occur in approximately 25% of diffuse large B-cell lymphoma (DLBCL) cases, including approximately 30% of activated B-cell DLBCL and up to 70% of primary extranodal DLBCL, and are associated with poor survival following 1st line therapy. Furthermore, based on new genetic classifications, co-mutations in MYD88 and CD79 (C5 and MCD subgroups) are associated with an inferior survival after standard R-CHOP. MYD88 mutations result in activation of the NF-κB pathway which drive a range of pro-tumor activities, including upregulation of proinflammatory cytokines and genes involved in tumor cell proliferation and survival. Activation of this pathway results in upregulation of IRF4 through the transcription factors Ikaros and Aiolos, which in turn further augments NF-kB activation while simultaneously downregulating Type I IFN signaling, thereby preventing oncogene-induced cell death. Constitutive NF-kB pathway activation resulting from MYD88 mutations is dependent on the interleukin-1 receptor associated kinase 4 (IRAK4), a key component of the myddosome complex which normally stimulates NF-kB signaling following TLR or IL-1R engagement and MYD88 activation. KT-413 is a potent and selective heterobifunctional small molecule protein degrader mediating the degradation of both IRAK4 and the IMiD substrates Ikaros and Aiolos via the ubiquitin-proteasome system. The therapeutic hypothesis is that degradation of IRAK4 and IMiD substrates will maximize NF-κB inhibition while simultaneously upregulating the Type I Interferon response, thus restoring the apoptotic response and enabling oncogene-mediated cell death, resulting in robust antitumor response in MYD88-mutant DLBCL. KT-413 induced strong antitumor activity, including complete or partial regressions, in the in-vivo preclinical models of MYD88MT DLBCL and showed strong tumor growth inhibition in MYD88MT patient-derivedxenograft models (Mayo 2021). Methods: KT-413 is being evaluated in an open label, dose escalation (Phase 1a) study in patients with relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (NHL) according to the 2016 World Health Organization (WHO) classification, followed by dose expansion (Phase 1b) in patients with R/R DLBCL with documented tumor MYD88 mutation status. All patients must have at least one bi-dimensionally measurable disease site per Lugano Classification and be relapsed and/or refractory to at least 2 accepted standard systemic regimens and ineligible or refused autologous stem cell transplant (ASCT) or CAR-T therapy; however, prior ASCT or CAR-T therapy are not exclusionary. Patients with known CNS lymphoma or meningeal involvement are excluded as well as those that received prior treatment with an IRAK4 inhibitor, experienced disease progression on IMiD-containing regimen < 6 months, are R/R after ≥ 2 prior IMiD-containing regimens or discontinued a prior IMiD therapy due to IMiD-related toxicity. Patients who underwent a prior allogeneic hematopoietic stem cell transplant are also excluded. The Phase 1a (n=40) utilizes an accelerated titration followed by a 3+3 design in ascending doses of IV administered KT-413 in once every 21-day cycles to identify the maximum tolerated dose (MTD)/recommended Phase 2 dose (RP2D) (primary endpoint). Secondary and exploratory endpoints include pharmacokinetics (PK) and preliminary pharmacodynamic effects (PD) using blood and tumor tissue, respectively. Once a tolerable and pharmacologically optimal dose is determined in 3-6 patients, it will be confirmed by enrolling additional patients with B-cell NHL, for a total of nine patients. In Phase 1b, up to 40 R/R DLBCL patients will be enrolled into one of two cohorts (n=20): MYD88MT or MYD88WT and treated at the RP2D to further characterize safety, tolerability, preliminary efficacy, PK, and PD. Enrollment in the Phase 1a portion of the KT413-DL-101 study is ongoing. NCT05233033.

DA-R-EPOCH versus R-CHOP in intermediate and high risk IPI diffuse large B-cell lymphoma, a randomized controlled trial

Background: Diffuse large B-cell lymphoma is a challenging disease in management. Although most patients are cured with the standard R-CHOP, one third of them remains refractory to this regimen. As prognosis of refractory disease is worse than primarily responding one, several trials investigated other more intense frontline regimens tailored based upon risk and biological characteristics. Patients &amp; Methodology: This is a prospective randomized trial investigating the more intensive DA-R-EPOCH regimen as frontline therapy in intermediate and high risk DLBCL patients in comparison to the standard R-CHOP regimen. We compared both regimens in these risk categories as well as undergoing a subgroup analysis according to cell of origin (Germinal center versus activated B cell) and according to BCL2 and C-myc expression (double expressor lymphoma). Toxicities in both arms have been analyzed according to common terminology criteria for adverse events (CTCAE). Results: In spite of being more toxic and complex, there was no significant improvement in DFS or response rate with DA-R-EPOCH. No significant benefit for DA-R-EPOCH over R-CHOP in both germinal center and activated B-cell DLBCL. Conclusion: Tailoring upfront treatment of DLBCL based upon risk classification or BCL2/c-myc expression remains an area of unanswered questions and warrants further investigations.

P1267: HSP110 FAVORS BCR SIGNALING THROUGH SYK PHOSPHORYLATION IN DIFFUSE LARGE B CELL LYMPHOMA

Background: Heat-shock proteins (HSPs) are molecular chaperones highly expressed in hematological malignancies. They promote proliferation, immune escape, and resistance to chemotherapies. Our team and others identified HSP110 as a pro-survival factor in diffuse large Bcell lymphomas (DLBCL). In particular, HSP110 sustains aberrant NFkB signaling in activated B-cell DLBCL (ABC-DLBCL) through MyD88 stabilization (Boudesco et al. Blood 2018 Aug 2;132(5):510-520). These findings highlighted HSP110 as a new potential therapeutic target in DLBCL. However, the role of HSP110 in the global signaling pathway of DLBCL has not been studied yet. In addition, the impact of HSP110 inhibition in in vivo xenograft models has been hampered by the absence of specific inhibitors. Aims: We recently identified two foldamers by their capacity to specifically disrupt HSP110 functions in colorectal cancer models (Gozzi et al. Cell Death Differ. 2020 Jan;27(1):117-129). These first HSP110 inhibitors have given us the opportunity to gain more insight into the signaling pathways controlled by HSP110 in DLBCL in vitro and in vivo. Methods: We treated DLBCL cell lines in vitro, and in vivo in xenografted NSG mice. Analyses were done using Western blot, IHC, proximity ligation assay, and in vitro phosphorylation assay. Results: We first validated the two HSP110 inhibitors in DLCBL since they efficiently disrupted the binding of HSP110 to MyD88 in DLBCL cell lines. They impaired cell growth and led to cell death in the Myd88L265P cell lines OCI-Ly10, HBL1, TMD8 and OCI-Ly3 in vitro. Treatment of TMD8-xenografted NSG mice with increasing doses of HSP110 inhibitors limited tumor growth, without tissue toxicity. Analysis of xenografted tumors and in vitro treatments of DLCBL cell lines revealed that the MyD88/NFkB pathway was altered as expected, but also the BCR pathway since we observed a decrease in the activation of BTK, SYK and PLCy2. Using immunoprecipitations and DuolinkTM assays, we identified an in vitro and in cellulo physical interaction between HSP110 and SYK (but not BTK) in several DLBCL cell lines. Furthermore, HSP110 inhibitors disrupted the binding of HSP110 with SYK in vitro, suggesting that the foldamers may act through this mechanism in vivo. We confirmed this assumption using DuolinkTM assays, which showed fewer interactions in xenografted tumors after treatment. Moreover, the phosphorylation of SYK recombinant protein is increased in vitro in the presence of a HSP110 recombinant protein, suggesting that HSP110 acts as a molecular chaperone for optimal activation of SYK through phosphorylation. In addition to the MyD88L265P promotion of the NFkB pathway, the chronic BCR activation in DLBCL has been shown to mediate the activation of both the SYK/BTK and PI3K/Akt pathways, converging to NFkB. Targeting these pathways with ibrutinib and copanlisib, respectively, synergistically suppresses tumor growth in xenograft models (Paul et al. Cancer Cell 31, 64–78, January 9, 2017). We showed that the combination of HSP110 inhibitors with copanlisib also decreases SYK/BTK and AKT phosphorylation synergistically, leading to less NFkB activation in vitro and in vivo. Accordingly, the drug combination stopped the progression of the xenograft. Summary/Conclusion: In conclusion, we identified HSP110 as a regulator of BCR signaling through SYK chaperoning in DLBCL. This finding highlights the major potential of HSP110 as a therapeutic target in DLBCL and suggests a broader role in immune responses.