Protein arginine methyltransferase 5 (PRMT5), a type II arginine methyltransferase, is overexpressed in several aggressive B-cell malignancies and facilitates cancer cell proliferation. JNJ-64619178, a selective small-molecule inhibitor targeting PRMT5, has previously shown promising preclinical activity across a range of hematological malignancies; however, the clinical activity of JNJ-64619178 monotherapy is limited despite strong target engagement. Therefore, we sought to identify rational combination partners for JNJ-64619178 to achieve improved activity in B-cell malignancies. Using dynamic Bcl-2 homology 3 (BH3) profiling, a functional assay to evaluate the net increase in proapoptotic signaling in response to drugs, we found that JNJ-64619178 increased overall proximity to apoptotic cell death (mitochondrial apoptotic priming) and dependence on B-cell leukemia/lymphoma (BCL)-2 for survival (BCL-2 dependence), particularly in diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cell lines. In other B-cell non-Hodgkin lymphoma (B-NHL) cell lines that are primarily MCL-1 dependent and less BCL-2 dependent, JNJ-64619178 increased mitochondrial apoptotic priming without shifting anti-apoptotic dependence from MCL-1 to BCL-2. Co-targeting PRMT5 and BCL-2 synergistically induced apoptosis in DLBCL and MCL cell lines that displayed at least partial BCL-2 dependence at baseline, but not in less BCL-2-dependent B-NHL cell lines. Interestingly, JNJ-64619178 upregulated death receptor 4 (DR4) and death receptor 5 (DR5) expression on the cell membrane of B-NHL cell lines, thereby sensitizing them, including the less BCL-2-dependent cell lines, to recombinant TRAIL-induced extrinsic apoptotic cell death. These findings highlight a role of PRMT5 in regulating both intrinsic and extrinsic apoptosis and suggest potential combination partners with PRMT5 inhibitors for potential clinical application in B-NHL.

Immune checkpoint blockade of CD47 has shown promising results in lymphoid malignancies, with its effects attributed to enabling tumor-cell phagocytosis. However, alternate cytotoxic cell death mechanisms have been reported, potentially contributing to the overall anti-tumor activity. Although previous studies have highlighted a mechanism of caspase-independent cell death, this mechanism has yet to be well-characterized, thereby warranting further investigation to comprehensively understand the anti-tumor mechanism of CD47 blockade to facilitate optimal drug partner selection for combination therapy. The fully humanized anti-CD47 monoclonal antibodies, SRF231, magrolimab, as well as a mouse monoclonal anti-CD47 antibody, B6H12, were used. Multiple cell death mechanisms were evaluated including apoptosis, autophagy and necroptosis by using customized Hoechst/Annexin V, the precision medicine technique BH3 profiling, as well as standard experimental techniques – flow cytometry, siRNA and CRISPR Cas9 genetic manipulation, Western blotting, and immunohistochemistry. These techniques were used on a comprehensive range of lymphoid malignant models including diffuse large B-cell lymphoma, Burkitt lymphoma, and T-acute lymphoblastic leukemia cell lines, patient primary chronic lymphocytic leukemia cells, as well as lymphoid cell-line derived and patient-derived xenograft mice, to elucidate the mechanism of cell death by CD47 blockade and to identify the optimal drug partners for treatment combination. We demonstrate that the anti-CD47 antibodies SRF231, magrolimab, and B6H12 eliminated tumor cells from various in vitro and in vivo lymphoid malignant models via the activation of the RIPK1/MLKL/necroptotic pathway. Moreover, the BH3 profiling technique distinguished two different lymphoid malignant models that respond differently to the BCL-2 inhibitor venetoclax when combined with SRF231; one highlighting the effective yet distinct mechanisms of SRF231-induced necroptosis and venetoclax-induced apoptosis in models that were specifically and/or highly dependent on BCL-2 for survival, while the other implicating venetoclax as a counterproductive partner with SRF231 in models that were not dependent on BCL-2 for survival or were not responsive to venetoclax treatment. Collectively, this study unravels a novel, non-canonical cell death mechanism of targeting CD47 by activating necroptosis, and provides evidence and rationale for further evaluation of a therapeutic strategy of combining CD47 blockade with and without apoptotic inducers for suitable patients with lymphoid malignancies.

Abstract Introduction: CD47 is a macrophage checkpoint protein that acts as a “don’t-eat-me” signal to prevent cell phagocytosis. Its blockade has shown promising results in clinical trials of lymphomas. Interestingly, CD47 blockade-induced cell death beyond phagocytosis, has also been reported, potentially contributing to the overall anti-tumor activity. This cell death mechanism has yet been well-characterized, thus warranting investigation to comprehensively unravel the mechanism of CD47 blockade and to facilitate the identification of optimal drug partners for combination therapy. Method: Anti-CD47 monoclonal antibodies (mAb), SRF231, magrolimab, B6H12, were evaluated for cell death mechanisms such as apoptosis, autophagy or necroptosis. Techniques used include BH3 profiling, Annexin V, siRNA/CrisprCas9, Western blot and immunohistochemistry. Diffused large B-cell lymphoma (DLBCL) and acute myeloid leukemia (AML) cell lines were used. In vitro results were used to select for appropriate drug to combine with CD47 blockade. Results were validated ex vivo in leukemia patient samples and in vivo in cell line and patient-derived mouse models. Result: Anti-CD47 mAbs consistently killed tumor cells from 10 cell lines, 24 patients, 3 mouse models by activating necroptosis, while sparing healthy immune cells. Necroptosis was confirmed via increased phospho(p)-RIPK and p-MLKL, which were rescued by necroptosis inhibitors or CD47/MLKL silencing. We further ascertained that PLCγ activation is upstream of necroptosis, as inhibiting PLCγ prevented p-MLKL and necroptosis. Moreover, apoptosis or autophagy was not involved, as inhibiting these pathways did not rescue SRF231-induced cell death. Given that necroptosis is the primary mechanism, we proceeded to leverage on apoptosis as an additional pathway to enhance cell death. Using BH3 profiling, a technique that informs cellular sensitivity to apoptotic inducers - BH3 mimetics, we identified the BCL-2 inhibitor venetoclax as an effective partner for SRF231 against hematologic malignant cells that depend highly on BCL-2 for survival (i.e. Cell survival: DMSO - 100%, (50nM) VEN - 71.39%, SRF231 - 57.01%, Combo - 19.53%, P < 0.0001). SRF231 and venetoclax combination completely eliminated tumor burden and prolonged progression free survival in BCL-2 dependent DLBCL and AML mouse models (i.e. Mice survival at day 70: Control - 0%, VEN - 0%, SRF231 - 58.3%, Combo - 100%; P < 0.0001). Importantly, SRF231 was equally effective against non-BCL-2 dependent, venetoclax-resistant DLBCL mouse models (i.e. Mice survival at day 200: Control - 0%, SRF231 - 62.5%, P < 0.0001). Conclusion: Our study unravels a novel cell death mechanism of CD47 blockade through necroptosis, thereby permitting the inclusion of venetoclax-induced apoptosis, a complementary combination worthy of further study against BCL-2 dependent hematologic malignancies in the clinic. Citation Format: Stephen Jun Chong, Rebecca Valentin, Jing Wang, Fen Zhu, Filip Garbicz, Kartini Iskandar, Brienne C. Toh, Marisa Peluso, Jeremy Zhang, Liam Hackett, Benjamin H. Lee, Li Ren Kong, Catherine J. Wu, Wee Joo Chng, Shazib Pervaiz, Carsten U. Niemann, Ruben D. Carrasco, Matthew S. Davids. CD47 blockade induces necroptosis and complements the effects of BCL-2 inhibition in hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5674.

Introduction: Immune checkpoint blockade of CD47, a macrophage checkpoint protein that serves as a “don't-eat-me” signal on cancer cells, is known to enable tumor-cell phagocytosis, and has shown promising results in clinical trials in lymphomas. However, additional cytotoxic cell death mechanisms beyond phagocytosis have also been reported, potentially contributing to the overall anti-tumor activity. The latter cell death mechanisms have yet to be well-characterized, therefore warranting further investigation to comprehensively understand the anti-tumor mechanisms of CD47-blockade in lymphoid malignancies to facilitate identification of optimal drug partners for combination therapy. Methodology: The fully humanized anti-CD47 monoclonal antibodies, SRF231, magrolimab, as well as the mouse monoclonal anti-CD47 antibody, B6H12, were evaluated in this study. Cell death mechanisms involving apoptosis, autophagy and necroptosis were analyzed using techniques such as BH3 profiling (Ryan, Biol Chem, 2016), Hoechst/Annexin V, flow cytometry, pharmacological and genetic (siRNA and CRISPR-Cas9 knockout) manipulations, Western blotting and immunohistochemistry. Cell lines including diffuse large B-cell lymphoma (DLBCL) (Ri-1, OCI-Ly1, TMD8, venetoclax-resistant OCI-Ly1-R), Burkitt's lymphoma (Raji), and T-acute lymphocytic leukemia (Jurkat) were used for in vitro analyses. Results generated from in vitro experiments were used to select promising drugs for treatment combination with CD47 blockade. Findings were validated using primary, peripheral blood-derived chronic lymphocytic leukemia (CLL) cells, purified human monocytes, T and B cells, treated ex vivo as well as in vivo in various lymphoid cell line-derived (CDX) and DLBCL patient-derived xenograft (PDX) mouse models. Results: The anti-CD47 antibody SRF231 consistently killed tumor cells in lymphoid malignant cell lines, primary CLL cells, CDX and PDX models by activating necroptosis, while sparing other immune cells such as monocytes, T and B cells. The induction of necroptosis was confirmed by demonstrating increased phospho-activation of RIPK and downstream phospho-MLKL, which were abrogated by multiple pharmacological inhibitors of necroptosis and genetic silencing/knockout of CD47, RIPK and/or MLKL. We further ascertained that this necroptotic programmed cell death did not involve apoptotic- or autophagic-cell death, as caspase cleavage or LC3-flux were absent, respectively. Moreover, inhibiting apoptosis with Z-VAD-FMK or autophagy with BAPTA-AM did not rescue SRF231-induced cell death. Given that necroptosis was the primary cell death mechanism, we utilized BH3 profiling technique to identify a potential combination partner to specifically induce apoptosis in our lymphoid malignant models, to potentially leverage an additional cell death pathway to enhance tumor cell death. BH3 profiling identified the BCL-2 inhibitor venetoclax as a potentially effective therapeutic partner for SRF231 against lymphoid malignant cells that are specifically and highly dependent on BCL-2 for survival (Survival rate: DMSO – 100%, (50nM) VEN – 71.39%, SRF231 – 57.01%, Combo – 19.53%, P-value < 0.0001). A treatment combination with SRF231 plus venetoclax in vivo completely eliminated tumor burden and prolonged progression-free survival in a BCL-2-dependent lymphoma xenograft model (Mice survival rate at day 70: Isotype control – 0%, VEN – 0%, SRF231 – 58.3%, Combo – 100%; P-value < 0.0001). Importantly, SRF231 was equally effective against non-BCL-2 dependent, venetoclax-resistant lymphoma models (PDX mice survival rate at day 200: Isotype control – 0%, SRF231 – 62.5%, P-value < 0.0001) . Conclusion: Our study unravels a novel, critical non-canonical cell death mechanism of targeting CD47 via necroptosis. We also demonstrate the complementary and distinct cell death mechanisms of SRF231-induced necroptosis and venetoclax-induced apoptosis, a combination worthy of further study in the clinic specifically against BCL-2-dependent lymphoid malignancies.

Intratumor heterogeneity impacts disease progression and therapeutic resistance but remains poorly characterized by conventional histologic, immunophenotypic, and molecular approaches. Single-cell biophysical properties distinguish functional phenotypes complementary to these approaches, providing additional insight into cellular diversity. Here, we link both buoyant mass and stiffness to gene expression to identify clinically relevant phenotypes within primary mantle cell lymphoma (MCL) cells, using MCL as a model of biological and clinical diversity in human cancer. Linked measurements reveal that buoyant mass and stiffness characterize B cell development states from naïve to plasma cell and correlate with expression of oncogenic B cell receptor signaling genes such as BLK and CD79A . In addition, changes in cell buoyant mass within primary patient specimens ex vivo correlate with sensitivity to Bruton’s tyrosine kinase inhibitors in vivo in MCL and chronic lymphocytic leukemia, another B cell malignancy. These findings highlight the value of biophysical properties as biomarkers of response in pursuit of future precision therapeutic strategies.

Introduction: Protein arginine methyltransferase 5 (PRMT5), a type II arginine methyltransferase, is overexpressed in several aggressive B cell malignancies and facilitates cancer cell growth. JNJ-64619178 (JNJ-9178) is a PRMT5-selective small molecule inhibitor that has been investigated in a clinical trial in patients with advanced solid tumors, B-cell non-Hodgkin lymphoma (NHL) and lower risk myelodysplastic syndromes [NCT03573310]. However, clinical activity was limited despite robust target engagement. Here, we investigated the effects of JNJ-9178 on both the intrinsic and extrinsic apoptosis pathways and identified rational combination partners for JNJ-9178 to achieve improved activity in B-cell NHL. Methods: Dynamic BH3 profiling (DBP) was employed to quantify the net pro-apoptotic signaling triggered by ex vivo drug treatments. Other standard techniques include gene knockdown via shRNA, Western blotting, PI/Annexin V and FACS analysis. Various cell lines, including DLBCL (TMD8, Ri-1, OCI-Ly1, OCI-Ly1R, SUDHL4), double-hit lymphoma patient-derived xenograft (DW19), MCL (Mino, Jeko-1), and BL (Raji, BL-70) were utilized to investigate the in vitro anti-cancer properties of JNJ-9178, BH3 mimetics (venetoclax [Ven, BCL-2i], S63845 [S63, MCL-1i], A1331852 [A133, BCL-xLi]) and TRAIL analogs (rhTRAIL [recombinant human TRAIL], Conatumumab [DR5 agonist], and Mapatumumab [DR4 agonist]). Drug synergism was calculated using SynergyFinder. A luciferase-labeled OCI-Ly1 cell line-derived xenograft (CDX) mouse model was employed for in vivo drug evaluation. Results: BH3 profiling in a panel of B-cell NHL cell lines identified 7 partially BCL-2-dependent cell lines (DW19, TMD8, Ri-1, SUDHL4, OCI-Ly1, Jeko-1 and Mino). These cell lines were not sensitive to Ven (except for OCI-Ly1 and Ri-1) due to co-dependence on other anti-apoptotic proteins. DBP demonstrated that JNJ-9178 exposure increased overall mitochondrial apoptotic priming and further increased BCL-2 dependence in all 7 cell lines. The combination of JNJ-9178 and Ven synergistically induced apoptosis in all 7 cell lines. We also identified 3 less BCL-2-dependent cell lines, Raji (BCL-2 sufficient), BL-70 (BCL-2 deficient) and OCI-Ly1R (with Ven-acquired resistance generated from the OCI-Ly1 cell line). All 3 cell lines were primarily MCL-1 dependent and sensitive to S63 but resistant to Ven. DBP in these cell lines demonstrated that JNJ-9178 increased overall mitochondrial apoptotic priming and MCL-1 dependence while having negligible impact on BCL-2 dependence. Consistently, the cells became more sensitive to S63-induced apoptosis in the presence of JNJ-9178 while remaining resistant to Ven. Together, these data suggest that JNJ-9178 increases overall mitochondrial apoptotic priming without switching BCL-2-less dependent cell lines to become BCL-2-dependent. In a CDX mouse model, we found the combination of JNJ-9178 with Ven significantly delayed tumor growth and prolonged survival compared to single drug treatment. Examining death receptor expression on the cell membrane via flow cytometry, we found that JNJ-9178 led to increased membrane expression of the TRAIL receptors DR4 and DR5 in B-cell NHL cell lines including DLBCL, MCL and BL, and sensitized these cells to rhTRAIL-induced apoptosis. Utilizing targeted knockdown and the agonist antibodies Conatumumab and Mapatumumab, we found the synergism between JNJ-9178 and rhTRAIL required activation of both DR4 and DR5 in most cell lines. Mechanistically, the increased expression of membrane DR4 and DR5 by JNJ-9178 could be due to a cellular response to global splicing alterations as we found that splicing modulators targeting different components of the splicing machinery other than SmB, SmD1 and SmD3 also led to increased membrane DR4 and DR5 expression and sensitized tumor cells to rhTRAIL-induced apoptosis. We tested the combination of JNJ-9178 with conatumumab in the CDX mouse model and found this combination significantly delayed tumor growth and prolonged survival compared to single drug treatment. Conclusions: We identified PRMT5 as an important regulator of both intrinsic and extrinsic apoptosis. Our data suggest that DBP has the potential to optimize the selection of BH3 mimetics to combine with JNJ-9178 to maximize the activity of this drug across certain B-cell NHL subtypes. Additionally, JNJ-9178 sensitizes B-cell NHL cell lines to TRAIL-induced cancer cell-selective extrinsic apoptosis.

ABSTRACT:The development of targeted therapy for patients with multiple myeloma (MM) is hampered by the low frequency of actionable genetic abnormalities. Gain or amplification of chromosome 1q (1q+) is the most frequent arm-level copy number gain in patients with MM and is associated with higher risk of progression and death despite recent therapeutic advances. Thus, developing targeted therapy for patients with MM with 1q+ stands to benefit a large portion of patients in need of more effective management. Here, we used large-scale dependency screens and drug screens to systematically characterize the therapeutic vulnerabilities of MM with 1q+ and displayed increased sensitivity to myeloid cell leukemia-1 (MCL1) and phosphatidyl inositol 3-kinase (PI3K) inhibitors. Using single-cell RNA sequencing, we compared subclones with and without 1q+ within the same patient tumors and demonstrated that 1q+ is associated with higher levels of MCL1 and the PI3K pathway. Furthermore, by isolating isogenic clones with different copy number profiles for part of the chromosome 1q arm, we observed increased sensitivity to MCL1 and PI3K inhibitors with arm-level gain. Lastly, we demonstrated synergy between MCL1 and PI3K inhibitors and dissected their mechanism of action in MM with 1q+, uncovering a cytostatic effect. In conclusion, this study highlights that MM with 1q+ may present enhanced sensitivity to MCL1 and PI3K inhibitors, enabling their use at lower doses without sacrificing efficacy, and may thus accelerate the development of targeted therapy for patients with MM and 1q+.

Introduction: PRMT5, a type II arginine methyltransferase, is overexpressed in several aggressive B cell malignancies and facilitates cancer cell growth. Small molecule inhibitors targeting PRMT5 show promising pre-clinical activity, particularly in mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). There have been a number of clinical trials evaluating PRMT5 inhibitors in patients with advanced solid tumors or non-Hodgkin lymphoma (NHL), revealing manageable dose-dependent toxicity and initial signs of anti-tumor activity in various tumor types [NCT06137144, NCT05528055, and NCT03573310]. The BCL-2 inhibitor venetoclax demonstrates promising monotherapy activity in NHL. Here, we explore the mechanisms underlying the therapeutic potential of PRMT5i-1 (P1), a SAM and ATP competitive PRMT5 inhibitor, in combination with venetoclax in B cell malignancies. Methods: Dynamic BH3 profiling (DBP) was employed to quantify the net pro-apoptotic signaling triggered by ex vivo drug treatments. Other standard techniques include RNA sequencing, gene knockdown via shRNA, immunoprecipitation (IP), Western blotting, PI/Annexin V and CellTiter-Glo. DLBCL (TMD8, RI-1, OCI-Ly1, SUDHL4), double-hit lymphoma (DHL) patient-derived xenograft (PDX) cell line (DW19), MCL (Mino, Jeko-1), and Burkitt lymphoma (Raji) cell lines were utilized to investigate the in vitro anti-cancer properties of P1 and BH3 mimetics (venetoclax [BCL2i], S63845 [S63, MCL-1i], A1331852 [A133, BCL-xLi], Selleckchem). Drug synergism was calculated using SynergyFinder, and a OCI-Ly1 cell line-derived xenograft (CDX) mouse model was employed for in vivo drug evaluation. Results: By utilizing a synergy score of 10 as a threshold, combining P1 with venetoclax synergistically increased apoptosis in all 8 cell lines tested with the exception of Raji cells. Interestingly, Raji cells were resistant to P1, venetoclax, and A133, but sensitive to S63. In contrast, the combination of P1 with S63 or A133 each led to synergistic apoptosis in only 4 of the cell lines tested (S63: DW19, TMD8, OCI-Ly1, Mino; A133: DW19, OCI-Ly1, Jeko, Mino). Therefore, venetoclax appears to be a more effective partner for P1 compared to S63 and A133. Knockdown of PRMT5 also increased the sensitivity of cells to venetoclax. In a CDX mouse model, combining P1 with venetoclax significantly delayed tumor growth and prolonged mouse survival compared to single-drug treatment. Through DBP analysis, P1 was found to enhance the overall mitochondrial apoptotic priming of DLBCL and MCL cell lines (>15% increase in cytochrome c loss). Notably, Raji cells exhibited lower basal levels of apoptosis priming, and P1 had minimal impact on apoptotic priming in this particular cell line. To elucidate the mechanism behind the synergy between P1 and venetoclax, we performed RNA sequencing analysis on P1 treated OCI-Ly1 and SUDHL4 cell lines and found a significant (Q value< 0.001) enrichment of genes in several pathways, including alternative splicing and apoptosis. However, the protein expression of BCL-2 family members had minimal alterations upon P1 treatment. Subsequently, through IP we observed an enhanced interaction between BCL-2 and BIM following P1 treatment, potentially contributing to the heightened sensitivity to venetoclax. To determine if the combination of P1 and venetoclax activates cell death other than mitochondrial apoptosis, BAX and BAK double knockdown (DKD) cell lines were created and tested for their sensitivities to the two drugs. DKD cells were partially protected from venetoclax and P1 induced cell apoptosis. Furthermore, the pan-caspase inhibitor Z-VAD-FMK rescued the drug combination-induced cell death, indicating the involvement of caspases. The caspase 8 inhibitor Z-IETD-FMK also rescued drug-induced cell death, but not as much as Z-VAD-FMK, suggesting potential activation of the extrinsic apoptosis pathway by the combined use of P1 and venetoclax. Conclusions: Our study suggests that the combination of a PRMT5 inhibitor P1 with venetoclax potently induces both intrinsic and extrinsic apoptotic cell death and may serve as a potential therapeutic strategy to explore further for DLBCL and MCL.

Background The phase (ph) 3 AMPLIFY study for patients (pts) with treatment-naïve (TN) CLL will provide data on a fixed-duration acalabrutinib (A), venetoclax (V), obinutuzumab (O) triplet; however, AMPLIFY excluded pts with TP53 aberrant CLL. In the initial paper on our ph2 trial of an MRD-guided AVO triplet regimen in all-comers with TN CLL (Davids et al., Lancet Onc, 2021), we reported high rates of CR and uMRD irrespective of TP53 status. We therefore added an expansion cohort restricted to pts with TP53 aberrancy. Here, we report the primary endpoint evaluation for the entire study. Methods All-comer pts with TN CLL were initially enrolled in this investigator-sponsored, ph2 study (cohort 1), followed by a multi-center expansion restricted to TN pts with TP53-aberrant CLL (cohort 2) (NCT03580928). Pts received one 28-day cycle (C) of A 100 mg bid, followed by 2 Cs of AO (O at standard dosing), with V then added with an abbreviated 4 week ramp-up: C4D1 20 mg, C4D2 50mg, weekly ramp-up thereafter to 400 mg QD. AVO was given C4-7, then AV C8-15. If the primary endpoint of BM-uMRD (by flow at 10-4) with CR was reached at C16D1, pts discontinued (dc'd) therapy (tx); if not, pts continued AV through C24 and dc'd if BM-uMRD at C25D1. Pts who dc'd and later developed recurrent MRD could restart AV. Pts with detectable BM-MRD after C24 continued AV indefinitely. Efficacy was assessed by 2018 iwCLL criteria, toxicities by CTCAE v5, MRD by central 8-color flow and NGS clonoSEQ. BH3 profiling was performed as previously described (Ryan J et al., Biol Chem, 2016) on PB samples at screening and after C3. Results The study is fully enrolled with 72 pts (cohort 1 all-comers: n=37 (including 10 TP53-aberrant), cohort 2 TP53-aberrant: n=35). Median age 63 yrs (range 36-80); 68% male; 63% TP53-aberrant (del[17p] or TP53 mut); 17% NOTCH1 mutation, 28% complex karyotype (≥3 abnormalities); 75% unmutated IGHV (uIGHV). As of April 10, 2024, median follow-up was 54.8 mo. (cohort 1, 60.9 mo, cohort 2, 38.5 mo), and median number of cycles given was 25 (range 3-70). The primary endpoint of BM-uMRD CR at C16D1 was achieved in 42% (30/72), with no difference for pts with TP53 aberration at 42% (19/45). The best ORR was 98.6% (including 61% CR). C16 CR rates for mIGHV and uIGHV pts were 60% and 44%. At C16D1, MRD by flow at 10-4 in pts without and with TP53 aberration was: PB-uMRD 85%, 71% and BM-uMRD 89%, 71%, and in pts with mIGHV and uIGHV: PB-uMRD: 67%, 80% and BM-uMRD: 73%, 78%. Rates of best PB- and BM-uMRD overall were 94% and 85%. At C16, PB-uMRD by NGS at 10-5 was 56% (37/66, with 6 pts not tested), with no difference based on TP53 status. 79% (57/72) of pts with BM-uMRD electively d/c'd tx (39% and 40% after C15 and C24), 11 pts remain on active tx, and 4 pts d/c'd prior to C16 (2 withdrew consent, 1 non-compliance, 1 Gr5 COVID-19). Median time off tx for pts who d/c'd is 27.5 mo (range 1-50). 7 pts have progressed (3 CLL, 4 transformation (2 DLBCL, 2 Hodgkin), and 2 pts have died (Gr5 COVID-19 while on tx and DLBCL Richter's). 5 pts have started re-treatment with AV, with 3 achieving response (including 2 CR), 1 not yet evaluated, and one with Richter soon after re-starting. 4-yr PFS/OS for the whole study population, uIGHV, and TP53 aberrant was 89%/99%, 87%/95%, 77%/92%. In all 72 pts, all-grade heme tox included: neutropenia (73%; 37% Gr3/4) and thrombocytopenia (72%; 29% Gr3/4). Selected non-heme tox: headache (76%, 63% Gr1, 11% Gr2, 1% Gr3), bruising (66%, 65% Gr1, 1% Gr2), diarrhea (46%; 34% Gr1, 7% Gr2, 6% Gr3), infection (46%, 9.7% Gr3, 1 Gr5 COVID-19), hypertension (34%; 10% Gr3), infusion-related reaction (28%, 4% Gr3), arthralgia (30%, 27% Gr1, 1.5% Gr2, 1.5% Gr3), and any grade afib 4 pts (5.6%). Non-skin second malignancies occurred in 6 pts (8.3%), including 1 MDS. 18 pts (25%) had dose-reduction of only A (4.2%), only V (8.3%), or both drugs (12.5%). BH3 profiling in 53 pts with available serial samples found no difference in apoptotic priming based on TP53 or IGHV status, consistent with the similar efficacy of AVO in higher risk pts. Increased dependence on BCL-2 was observed in pts who achieved uMRD at C16. Conclusion In pts with TP53-aberrant CLL, the AVO triplet is a well-tolerated and highly active MRD-guided frontline therapy, with 71% of pts achieving PB and BM-uMRD (flow at 10-4) at C16. Responses are durable, with 89%, 87%, and 77% 4-yr PFS in all-comer, uIGHV, and TP53 aberrant pts. Our data support further study of AVO for pts with TN high risk CLL in GCLLSG CLL16 and future trials.

Despite recent advances in the therapy of diffuse large B-cell lymphoma (DLBCL), many patients are still not cured. Therefore, new therapeutic strategies are needed. The anti-apoptotic B-cell lymphoma 2 (BCL2) gene is commonly dysregulated in DLBCL due to various mechanisms such as chromosomal translocation t(14;18)(q32;q21) and copy number alterations; however, targeting BCL-2 with the selective inhibitor, venetoclax, led to response in only a minority of patients. Thus, we sought to identify a rational combination partner of venetoclax to improve its activity against DLBCL cells. Utilizing a functional assay, dynamic BH3 profiling, we found that the DNA hypomethylating agent decitabine increased mitochondrial apoptotic priming and BCL-2 dependence in DLBCL cells. RNA-sequencing analysis revealed that decitabine suppressed the pro-survival PI3K-AKT pathway and altered the mitochondria membrane composition in DLBCL cell lines. Additionally, it induced a DNA damage response and increased BAX and BAK activities. The combination of decitabine and venetoclax synergistically suppressed proliferation of DLBCL cells both in vitro and in vivo in a DLBCL cell line-derived xenograft mouse model. Our study suggests that decitabine plus venetoclax is a promising combination to explore clinically in DLBCL.

Hyperactivity of the cysteine protease cathepsin S (CTSS) -either through Y132 mutations or amplification/overexpression- is a recurrent alteration in follicular lymphoma (FL) and promotes tumor growth by inducing a supportive immune microenvironment (Bararia et al, 2020). Of note, patients with CTSS-hyperactive FL had better outcomes with standard therapies, suggesting that CTSS-hyperactivity can sensitize tumors to treatment. CTSS hyperactivity has also been reported in other B cell lymphomas (BCLs) (Dheilly et al, 2020) and solid cancers (Olson & Joyce, 2015). CTSS is mainly localized intralysosomally but can be released into the cytosol by lysosomal membrane permeabilization (LMP). Low level LMP can occur spontaneously (e.g., during cell division) and can be enhanced by treatment. Unlike other cathepsins, cytosolically released CTSS maintains its enzymatic activity at non-acidic pH. Thus, we aimed to (i) identify the determinants of the cytosolic CTSS activity, (ii) determine its impact on the regulation of apoptosis, and (iii) study LMP as a therapeutic approach for CTSS-hyperactive tumors. First, we accrued biochemical, functional, and clinical data supporting the role of cystatin B (CSTB) as a clinically relevant endogenous CTSS inhibitor in BCLs. Through unbiased and complementary proteomics (BioID2 labelling and co-IP followed by LC-MS/MS) we identified CSTB as a direct CTSS-interacting protein (8-fold enriched in the BCL cell line Karpas422 engineered to express CTSS wild type (WT) or Y132D vs CTSS knock-out (KO), P=0.0002). Single-cell RNA-Seq of primary FL (N=10) showed significantly higher CSTB expression in FL cells compared to normal B cells ( P=0.004). Moreover, shRNA mediated knock-down (k/d) of CSTB increased the overall cathepsin activity in BCL cell lines (N=8) by 1.5 to 5.5-fold, most notably in CTSS-hyperactive cells ( Fig A, top). We next employed LMP-inducing tool compounds (LLOMe) and clinically used drugs or analogs (desipramine, hexamethylene amiloride) to release cathepsins into the cytosol. CTSS-hyperactive Karpas422 were significantly more sensitive to LMP-inducing treatments compared to native cells (1.5 to 10-fold reduction of IC50). Importantly, CTSS hyperactivity and CSTB k/d increased LMP-mediated cell killing ( Fig A, bottom). Thus, the cytosolic CTSS/CSTB interaction determines the net cytosolic cathepsin activity and sensitivity of cells to undergo LMP-induced cell death. Next, we hypothesized that LMP-induced cytosolic CTSS hyperactivity could prime BCLs towards apoptosis. We used BH3 profiling to functionally quantify the dependencies and interactions of BCL2 family members in BCLs with and without CTSS hyperactivity. In Karpas422 cells expressing CTSS Y132D, LMP increased overall apoptotic priming and dependencies on the anti-apoptotic proteins MCL-1 (delta priming >30 % at 10 µM, P=0.04), BCL-xL (>45 % at 10 µM, P=0.0006) and BCL2 (> 45 % at 0.5 and 1 µM, P=0.0001). We hypothesized that BCL2 family members are proteolytically cleaved by cytosolic CTSS. Indeed, e.g., BCL2 protein levels were 2.5 to 3.5-fold lower in LLOMe-treated Karpas422 cells with CTSS-hyperactivity compared to CTSS KO, and CSTB k/d further decreased BCL2 levels. To validate CTSS-mediated cleavage of BCL2, we purified FLAG-tagged BCL2 and CTSS WT and Y132D. CTSS WT efficiently cleaved BCL2 in vitro <1 hour at the top ranked predicted cleavage site and the reaction rate increased 1.3-fold for CTSS Y132D. Finally, we hypothesized that LMP sensitizes cells to BCL2-targeting therapies ( Fig B). The combination of LLOMe-induced LMP and the BCL2 inhibitor venetoclax (VEN) showed increased cytotoxicity in CTSS-hyperactive Karpas422 cells compared to monotherapy and CSTB k/d enhanced this phenotype ( Fig A, bottom). We assessed cathepsin activities and generated dose-response curves for VEN with and without LLOMe-induced LMP in 15 primary CLL samples. Thereof, 12 samples had intermediate or high cathepsin activities and LLOMe-induced LMP increased their sensitivity to VEN, including a VEN-resistant sample in which the IC50 decreased to <3 nM. In summary, we show that CSTB is a functionally relevant inhibitor that determines the net activity of LMP-released cytosolic CTSS. Furthermore, LMP-inducing therapies may be a promising approach to sensitize CTSS-hyperactive tumors towards apoptosis by proteolytic cleavage of BCL2 family members.

This study investigated ibrutinib plus obinutuzumab in relapsed/refractory CLL, evaluating tolerability of 3 sequencing regimens as well as overall safety and efficacy. Fifty-two patients were initially randomized 1:1:1 to receive either obinutuzumab 1 month before ibrutinib initiation, ibrutinib 1 month prior to obinutuzumab initiation, or to start both drugs concomitantly. Higher rates of infusion-related reactions were observed with the first sequence, and only the latter 2 cohorts were expanded. Grade 4 hematologic toxicity was uncommon, and notable all-grade non-hematologic toxicities included bruising (58%), hypertension (46%), arthralgia (38%), diarrhea (37%), transaminitis (35%), atrial fibrillation (21%), and serious infection (17%). Best overall response rate was 96% (including 40% CR and 56% PR). Best rates of undetectable minimal residual disease in peripheral blood and bone marrow were 27% and 19%, respectively. With a median follow-up of 41.5 months, four-year progression-free and overall survival rates are 74% and 93%, respectively. Correlative studies demonstrated that serum CCL4 and CXCL13 levels were associated with clinical response, and BH3 profiling revealed increased BCL-2 and BCL-xL dependence in CLL cells from patients on treatment. Overall, ibrutinib plus obinutuzumab was highly active, with a manageable safety profile, supporting further investigation of this type of approach in relapsed/refractory CLL.

The development of targeted therapy for patients with Multiple Myeloma (MM) is hampered by the low frequency of actionable genetic abnormalities. Gain or amplification of chr1q (Amp1q) is the most frequent arm-level copy number gain in patients with MM, and it is associated with higher risk of progression and death despite recent advances in therapeutics. Thus, developing targeted therapy for patients with MM and Amp1q stands to benefit a large portion of patients in need of more effective management. Here, we employed large-scale dependency screens and drug screens to systematically characterize the therapeutic vulnerabilities of MM with Amp1q and showed increased sensitivity to the combination of MCL1 and PI3K inhibitors. Using single-cell RNA sequencing, we compared subclones with and without Amp1q within the same patient tumors and showed that Amp1q is associated with higher levels of MCL1 and the PI3K pathway. Furthermore, by isolating isogenic clones with different copy number for part of the chr1q arm, we showed increased sensitivity to MCL1 and PI3K inhibitors with arm-level gain. Lastly, we demonstrated synergy between MCL1 and PI3K inhibitors and dissected their mechanism of action in MM with Amp1q.

The B cell leukemia/lymphoma 2 (BCL-2) inhibitor venetoclax is effective in chronic lymphocytic leukemia (CLL); however, resistance may develop over time. Other lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax. Although genomic resistance mechanisms such as BCL2 mutations have been described, this probably only explains a subset of resistant cases. Using 2 complementary functional precision medicine techniques - BH3 profiling and high-throughput kinase activity mapping - we found that hyperphosphorylation of BCL-2 family proteins, including antiapoptotic myeloid leukemia 1 (MCL-1) and BCL-2 and proapoptotic BCL-2 agonist of cell death (BAD) and BCL-2 associated X, apoptosis regulator (BAX), underlies functional mechanisms of both intrinsic and acquired resistance to venetoclax in CLL and DLBCL. Additionally, we provide evidence that antiapoptotic BCL-2 family protein phosphorylation altered the apoptotic protein interactome, thereby changing the profile of functional dependence on these prosurvival proteins. Targeting BCL-2 family protein phosphorylation with phosphatase-activating drugs rewired these dependencies, thus restoring sensitivity to venetoclax in a panel of venetoclax-resistant lymphoid cell lines, a resistant mouse model, and in paired patient samples before venetoclax treatment and at the time of progression.

Background While venetoclax (V) plus obinutuzumab (O) is a highly effective frontline CLL regimen, outcomes remain suboptimal for high-risk patients (pts). The efficacy of ibrutinib plus VO in such pts is encouraging, but cardiac and infectious toxicities (tox) are common. We hypothesized that a time-limited triplet with the more selective BTKi acalabrutinib (A) would be active and well-tolerated. We previously published results on an initial cohort unrestricted by genetic risk (Davids et al., 2021), and now report on a new cohort with enrollment restricted to high-risk pts, as well as longer follow-up on the initial cohort. Methods Treatment (tx)-naïve pts with CLL with any genetic risk profile were initially enrolled in this investigator-sponsored, phase 2 study (cohort 1), followed by a multi-center expansion restricted to previously untreated pts with TP53-aberrant CLL (cohort 2). A starts at 100 mg BID for 28 days, followed by 2 cycles of AO (O at standard dosing), and V starts on C4D1 at 20 mg, then 50 mg on C4D2, with weekly ramp-up thereafter to 400 mg QD (total 4 week V ramp-up). AVO continues C5-7 (6 total cycles of O), and AV continues C8-15. If the primary endpoint of undetectable bone marrow MRD (BM-uMRD) CR is reached at C16D1, pts can discontinue therapy (tx); all others continue AV through C24, with the option to discontinue if BM-uMRD at C25D1. Assessments: efficacy by 2018 iwCLL criteria, toxicities (tox) by CTCAE v5, MRD by central 8-color flow (10-4 sensitivity) and NGS clonoSEQ (up to 10-6 sensitivity, Adaptive). BH3 profiling work is ongoing, performed as previously described (Ryan J et al., 2016) on peripheral blood (PB) samples after 1 and 3 cycles. Association of BCL-2 dependence with clinical response was compared by an unpaired t-test. Results As of 13 July 2022, 68 pts were enrolled (cohort 1 all-comers: n=37, cohort 2 TP53-aberrant: n=31) in this ongoing study (NCT03580928), with median follow-up of 35 mos (range 2-45). In all 68 pts, median age 63 yrs (range 36-80); 25% ≥70 yrs; 66% male; 60% TP53-aberrant (del(17p) or mut); 24% complex karyotype (≥3 abnormalities); 74% unmutated IGHV. Of the 56 pts evaluable to date at C16D1, 43% (24/56) achieved the primary endpoint BM-uMRD CR. The best ORR is 98% (48% CR, 50% PR). At C16D1, MRD by flow: 86% PB-uMRD, 86% BM-uMRD (Fig 1A). In a subset of 49 pts with available samples, 59% were PB-uMRD by NGS at 10-5 sensitivity (at 10-6, 59% of pts had indeterminate results due to low cell counts). In the 29 pts with TP53-aberrant disease evaluable at C16D1, the best ORR is 100% (52% CR, 48% PR), and 45% (13/29) achieved BM-uMRD CR, MRD by flow: 86% PB-uMRD, 83% BM-uMRD (Fig 1A). There were no differences in response or MRD based on IGHV status. 79% (19/24) of pts in BM-uMRD CR electively discontinued tx after 15 cycles; median time off tx for these pts is 22.9 mos (range 1-30). 2 of the 19 pts who discontinued tx have had MRD only recurrence, and 1 had CLL disease progression; all 3 restarted tx with AV and achieved PR. 3 pts developed Richter's syndrome: 1 with DLBCL after 15 mos on study, and 2 with Hodgkin transformation (one 13 mos after completing study tx and one 12 mos into study tx; both achieved CR to Hodgkin-directed tx). 93% of all pts (63/68) remain progression-free. In all 68 pts, all-grade heme tox included: neutropenia (75%; 37% Gr3/4), thrombocytopenia (73%; 28% Gr3/4), anemia (49%, 3% Gr3). Non-heme tox: headache (78%, 1% Gr3), fatigue (76%, 1% Gr3), bruising (66%, all Gr1/2), nausea (49%, all Gr1/2), diarrhea (40%, 4% Gr3), infusion-related reaction (30%, 4% Gr3), hypertension (27%; 9% Gr3), increased ALT (27%, 1% Gr3), arthralgia (25%, all Gr1), and infection (6% Gr3; 1 case of Gr5 COVID-19 pneumonia). 2.9% (2/68) pts had afib. 14 pts (21%) required dose-reduction of either only A (4%), only V (9%), or both drugs (7%). BH3 profiling is ongoing, and initial data suggest that pts who later went on to achieve CR may have a greater increase in BCL-2 dependence at C4D1 than pts who achieved PR as best response (p=0.05, Fig 1B). Conclusion AVO is a highly active, well-tolerated triplet in a frontline CLL population enriched for high-risk disease, with 83% of TP53-aberrant pts achieving BM-uMRD after 15 mos of tx. Responses are durable, with 93% PFS in all pts at a median follow-up of nearly 3 yrs. Low rates of cardiac and infectious tox were observed. Our data support continued investigation of the MRD-guided, time-limited AVO triplet. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Introduction: BCL2 family protein dysfunction is an important mediator of chemoresistance in lymphoid malignancies. Venetoclax (VEN), a BH3 mimetic that selectively targets the anti-apoptotic protein BCL2, is FDA approved for treatment of chronic lymphocytic leukemia (CLL), with clinical trials underway for other lymphoid malignancies. Despite the clinical efficacy of VEN monotherapy, complete remission and progression free rates are still relatively low, thereby indicating the existence of inherent or acquired mechanisms of resistance. Preclinically, increases in anti-apoptotic BCL2 and MCL1 phosphorylation (BCL2-P/MCL1-P) and MCL1 protein level have been implicated in chemoresistance. We therefore question if phosphorylation of these proteins may drive VEN resistance and if removing this modification may re-sensitize resistant cells to VEN induced cell death. Methodology: BH3 profiling (a functional assay that assesses mitochondrial apoptotic priming and anti-apoptotic protein dependence), western blot and cell death assays were performed. Result: We report that VEN acquired resistant DLBCL cell line (OCI-Ly1R) has increased BCL2-P, MCL1-P and MCL1 protein levels as compared to sensitive parental cell line (OCI-Ly1S), while inherently resistant DHL cell line (Su-DHL4) possesses higher MCL1-P and MCL1 protein levels as compared to OCI-Ly1S. We further show that this increase in phosphorylation status contributes to the induction of VEN resistance. Using BH3 profiling, we demonstrate that anti-apoptotic protein phosphorylation-driven VEN resistance involves a change in sensitivity to pro-apoptotic proteins, with an increase in sensitivity to MCL1 inhibition and reciprocal decrease in sensitivity to BCL2 inhibition. We further show that BCL2 and MCL1 dephosphorylation by PP2A activating drugs (PADs) re-sensitizes resistant cells to VEN treatment. Mechanistically, PADs such as the fingolimod (FTY720) not only dephosphorylate BCL2-P and MCL1-P, but also dissociate BAX from BCL2 and destabilize MCL1 protein. These changes lead to the rewiring of cellular sensitivity to BCL2 inhibition, thereby re-sensitizing both VEN acquired and inherent resistant cells to VEN induced cell death. Importantly, the PP2A activating effects of FTY720 were recapitulated in primary cells from the peripheral blood of 16 CLL patients, where FTY720 treatment also reduced BCL2-P, MCL1-P and MCL1 protein levels and dissociated BAX from BCL2. This led to an increased sensitivity to BCL2 inhibition and enhanced cell death upon combined treatment with FTY720 and VEN. Conclusion: Our work defines a new targetable mechanism of VEN resistance in lymphoid malignancies due to the phosphorylation of anti-apoptotic BCL2 family proteins. PADs re-sensitize resistant malignant cells to VEN treatment via dephosphorylation of anti-apoptotic proteins, suggesting a promising combination approach to explore further. Citation Format: Stephen J. Chong, Fen Zhu, Jolin X. Lai, Liam Hackett, Mary C. Collins, Shazib Pervaiz, Jean-Philippe Coppe, Matthew S. Davids. Targeting BCL2 family protein phosphorylation in venetoclax resistant lymphoid malignancies [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 3959.

Introduction: Protein Arginine Methyltransferase 5 (PRMT5) is a type II methyltransferase that catalyzes symmetric dimethylation of arginine. PRMT5 is highly expressed in several types of aggressive B cell malignancies, and increased PRMT5 expression has been associated with poor disease prognosis. Several small molecule inhibitors targeting PRMT5 have been developed and showed promising anti-tumor effect in pre-clinical studies including in mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). JNJ-64619178, a potent and specific PRMT5 inhibitor, exhibited broad antiproliferative activity in cancer cell lines from multiple cancer types and cell lines derived xenograft mouse model (Brehmer et al., 2021). In a phase 1 clinical trial of JNJ-64619178 (NCT03573310), patients with advanced solid tumors and non-Hodgkin lymphoma demonstrated manageable toxicity and preliminary evidence of antitumor activity at selected dose levels (Villar et al., 2020). In addition, in patients with lower-risk myelodysplastic syndromes, JNJ-64619178 had manageable hematologic toxicity at the selected expansion dose, but did not have significant clinical activity (Haque et al., 2021). Inhibition of PRMT5 alone could induce cell cycle arrest instead of apoptosis depending on the types of cancer (Zhu et al., 2019), therefore we hypothesized that the combination of PRMT5i and pro-apoptotic BH3 mimetics therapies could be synergistic in aggressive B cell malignancies. Methods: We used dynamic BH3 profiling (DBP) to measure the net pro-apoptotic signaling induced by ex vivo drug treatments (as per Montero et al., 2015). Other standard techniques included shRNA mediated gene knockdown, Western blot, cell viability assays including PI/Annexin V and CellTiter-Glo, and reactive oxygen species (ROS) measurement by flow cytometry with dihydroethidium (DHE) staining. The DLBCL cell lines (HBL1, TMD8, RI-1, OCI-Ly1, Karpas 422, SUDHL4, Toledo), double hit lymphoma (DHL) patient-derived xenograft (PDX) cell lines (DW19), mantle cell lymphoma (MCL) cell lines (Mino, Jeko-1), T cell acute leukemia (T-ALL) cell line (Jurkat) and Burkitt lymphoma (BL) cell line (Raji) were used to investigate the in vitro anti-cancer activity of JNJ-64619178 (Janssen Pharmaceuticals) and BH3 mimetics (venetoclax [BCL2i], S63845 [MCL-1i] and A1331852 [BCL-xLi], Selleckchem). SynergyFinder was used to calculate drug synergism. Results: In all 7 DLBCL cell lines and the DHL-PDX cell line, we found synergistic induction of apoptosis with JNJ-64619178 plus venetoclax (synergy score, 2.82~51.42). The lowest level of synergy was observed in OCI-Ly1 cells, which are intrinsically highly sensitive to venetoclax monotherapy. The combination of JNJ-64619178 with S63845 synergistically induced apoptosis in 5 out of 7 cell lines (synergy score, -1.91~35.64). While the combination of JNJ-64619178 with A1331852 synergistically induced apoptosis in all 7 cell lines (synergy score 1.88~46.73), 4 of them had weak synergy score (1.88~9.4). To confirm the effects we observed with pharmacologic inhibition of PRMT5, we performed shRNA mediated knockdown of PRMT5, which similarly revealed increased sensitivity to venetoclax. In MCL cell lines, combining JNJ-64619178 with venetoclax or A1331852 was more synergistic in inducing cell death than combining with S63845. The T-ALL Jurkat cell line was resistant to JNJ-64619178 and single BH3 mimetic-induced apoptosis. Combining JNJ-64619178 with A1331852 but not venetoclax or S63845 strongly induced apoptosis. The Burkitt cell line Raji was resistant to JNJ-64619178, venetoclax and A1331852, but was sensitive to S63845. The addition of JNJ-64619178 and a single BH3 mimetic in these Raji cells had only a minor synergistic effect. The synergy scores are summarized in Figure 1. Using DBP, JNJ-64619178 was found to increase overall apoptotic priming of DLBCL and MCL cell lines (> 15% increase in cytochrome c loss). By measuring ROS levels, we found increased ROS production (> 2-fold increase in median fluorescent intensity) in response to JNJ-64619178 before the onset of apoptosis, suggesting a possible mechanism by which JNJ-64619178 increases apoptotic priming. Conclusions: The combination of a PRMT5 inhibitor with BH3 mimetics, especially venetoclax, is worthy of further exploration as a potential therapeutic strategy for DLBCL and MCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background We recently discovered recurrent cathepsin S (CTSS) alterations, i.e., either somatic gain-of-function mutations (at the hotspot Y132) or amplification/overexpression of CTSS that result in aberrant hyperactivity of this lysosomal cysteine protease and promote tumor growth in follicular lymphoma (FL). In addition, CTSS is frequently overexpressed in other lymphomas and other cancers (e.g., Glioma, triple-negative breast cancer, renal cancer). Intriguingly, when released from the lysosome, CTSS is tightly linked with the regulation of cell death pathways. Materials and Methods BH3 profiling allows determining, to what extent a cell is primed towards cell death. It measures the extent of mitochondrial membrane permeabilization (MOMP) in response to BH3 peptides. Thus, BH3 profiling can record the net response of the pro- and anti-apoptotic interactions of the >15 apoptotic BCL2 family members in high-throughput (384-well plate flow-cytometry). Using BH3 profiling, we aimed to exploit the therapeutic vulnerabilities of CTSS hyperactive tumors, with a focus on compounds that induce lysosomal membrane permeabilization (LMP). Results Our preliminary data suggest that LMP is a promising novel therapeutic strategy in tumors with CTSS hyperactivity. Specifically, dose-response curves for cell viability indicated a higher sensitivity to LLOMe treatment in DG75 CTSS knock-out (CTSSKO) lymphoma cells with re-expression of CTSS WT (KO+CTSSWT), and highest sensitivity in DG75 with overexpression of CTSS Y132D (KO+CTSSY132D). Similar results were obtained in single-cell derived clones from Karpas422 lymphoma cells (CTSS KO vs WT vs Y132D). Release of cathepsins into the cytosol can lead to MOMP and apoptosis through the cleavage of BCL2 family members, including proteolytic inactivation of anti-apoptotic MCL1 and cleavage of BID which produces proapoptotic truncated BID (tBID). Of note, CTSS is the only cathepsin that remains enzymatically active at neutral pH for hours. Western blot confirmed increased cleavage of BID, MCL-1 and PARP-1 in LLOMe treated CTSSWT and CTSSY132D-cells, indicating that CTSS release by LMP can more efficiently induce apoptosis in CTSS hyperactive lymphomas. BH3 profiling confirmed that CTSS hyperactivity is associated with a dependency on MCL-1 and BCL-2 following LMP. In addition, CTSS hyperactive glioblastoma cell lines U-87 and U-251 also showed MCL-1 dependency following LMP, indicating that cytosolic CTSS hyperactivity primes for MCL-1 dependency across many cancer types. Conclusions Releasing CTSS from the lysosome is novel, attractive concept that can be combined with conventional, apoptosis-inducing therapies. Disclosure Information J. Hildebrand: None. P. Rengasayee: None. M. Antoniolli: None. L. Hackett: None. L. Adolph: None. S. Häbe: None. M. Heide: None. S. Wilhelm: None. C. Ludwig: None. C. Strobl: None. V. Passerini: None. M. Bergwelt-Baildon: None. R. Koch: None. M. Davids: None. O. Weigert: None.

Introduction: Despite recent advances in the therapy of diffuse large B cell lymphoma (DLBCL), many patients are still not cured, and therefore new therapeutic combinations are needed. The anti-apoptotic B cell lymphoma 2 (BCL-2) gene is commonly dysregulated in DLBCL due to various mechanisms such as chromosomal translocation t(14;18)(q32.3;q21.3), copy number alterations and gene amplifications; however, targeting BCL-2 with a selective inhibitor, venetoclax, led to response in only a minority of patients. Thus, we sought to identify a novel combination partner for venetoclax to improve response to therapy. Previously with dynamic BH3 profiling, we found that DNA hypomethylating agents (HMA) could increase BCL2 dependence in DLBCL cells. Here, we investigate the activity of the HMA decitabine with venetoclax in DLBCL. Methods: We utilized BH3 profiling, a functional assay to assess the mitochondrial priming of cells for apoptosis and the dependence of cells on various anti-apoptotic BCL-2 family proteins. Other standard techniques included Western blot, cell cycle (BrdU/7-AAD), CellTiter-Glo, and Seahorse XF cell mito stress test. The DLBCL cell lines (TMD8, HBL1, OCI-Ly3, OCI-Ly1, Karpas 422, SUDHL4, Toledo, OCI-Ly7) and double hit lymphoma (DHL) patient-derived xenograft cell lines were used to investigate the in vitro anti-cancer activity of decitabine and venetoclax. Results: Through BH3 profiling, we found heterogenous dependence of DLBCL, consistent with the variable expression of multiple anti-apoptotic proteins in DLBCL assessed by Western blot. DLBCL cells were less sensitive to single BH3 mimetic treatment (venetoclax, S63 (MCL-1i) and A133 (BCL-xLi)). Using dynamic BH3 profiling (which assesses the change in priming induced by ex vivo drug treatment), we found that decitabine primes DLBCL cells for apoptosis and increases BCL-2 dependence. Consequently, decitabine increases the sensitivity of DLBCL cells to venetoclax, as the combination of decitabine and venetoclax synergistically induces apoptosis and suppresses cell proliferation. Decitabine has previously been shown to increase TGF-β signaling in DLBCL by restoring the expression of a downstream target, SMAD1. Interestingly, we found that inhibition of TGF-β signaling only partially antagonizes the anti-cancer activity of decitabine, implicating other anti-cancer mechanisms of decitabine. Indeed, we found that decitabine also induces DNA damage and leads to cell cycle arrest in DLBCL cells. Furthermore, decitabine promotes activation of the key apoptotic effector proteins, BAK and BAX. Additionally, we found that this combination also suppresses oxidative phosphorylation, thus restricting the energy supply for DLBCL cells. Conclusions: The HMA decitabine sensitizes DLBCL cells to venetoclax in vitro through increasing apoptotic priming and BCL-2 dependence. This combination is worthy of further study in in vivo model systems. Citation Format: Matthew S. Davids, Fen Zhu, Stephen J. Chong, Jennifer Crombie, Liam Hackett, May C. Collins. Decitabine sensitizes diffuse large B cell lymphoma cells to venetoclax [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 5377.

Background: Combination ibr + obin was approved in frontline CLL based on results of iLLUMINATE; however, less data are available on this regimen in R/R CLL. Though active in R/R CLL, ibr monotherapy in RESONATE led to 4-year (yr) PFS/OS of ~45%/65%, leaving room for improvement. We previously reported initial safety and efficacy results of this phase 1b study investigating ibr + obin in R/R CLL (Davids et al., EHA 2020). Here, we report updated results, with median follow-up now of 41.5 months (mos). Aims: To assess longer-term safety and efficacy of ibr + obin in patients (pts) with R/R CLL. Methods: We conducted a phase 1b, investigator-initiated study of ibr + obin in R/R CLL (NCT02537613). The primary objective was to assess safety of 3 different drug sequencing regimens in cycle 1; the main secondary objective was to assess efficacy. Pts were randomized 1:1:1 to either receive obin 1 mo before ibr, ibr 1 mo before obin, or start concomitantly. Both drugs were given at the approved doses and schedule, with 6 total cycles of combination followed by ibr monotherapy until progression or unacceptable toxicity. Notable eligibility criteria: requiring treatment (tx) for R/R CLL/SLL by 2008 iwCLL criteria, ECOG PS ≤2, no prior ibr or obin. Assessments: toxicities by CTCAE v4, efficacy by 2008 iwCLL criteria, MRD by 4-8 color flow (10-4 sensitivity). Serum concentrations of 21 cytokines were assessed pre-tx and after 1 week of combination tx in a representative subset of 27 pts (n=9 per arm) by bead-based multiplex immunoassay (Eve Technologies, Calgary). Results: 52 pts were accrued, and all received ≥1 cycle of combination tx. Median age: 67 yrs (range 33-84); 77% male; median # prior tx: 1 (range 1-6); 25% del(17p) or TP53 mut; 27% del(11q); 50% unmutated IGHV; 27% bulky LAD (nodes ≥5 cm). As of 14 Feb 2022: median follow-up 41.5 mos (range 2.3-73.3). 27 pts (52%) remain on tx. All-grade heme toxicities: thrombocytopenia (88%; 23% Gr3/4), neutropenia (71%; 37% Gr3/4), febrile neutropenia (8%, all Gr3/4), anemia (73%; 8% Gr3). Notable non-heme toxicities: bruising (58%, all Gr1/2), arthralgia (38%, all Gr1/2), diarrhea (37%, all Gr1/2), transaminitis (35%; 4% Gr3), ≥Gr3 infection (17%, all Gr3), and cardiotoxicities: hypertension (46%; 10% Gr3), atrial fibrillation (21%; 10% Gr3). 1 pt had sudden cardiac death after 11 mos on study. 7 pts required ibr dose-reduction due to toxicity. The best ORR is 96%, including 50% CR and 46% PR (Fig. 1A). All 3 pts who previously progressed on venetoclax responded (1 CR, 2 PR). By ITT, 10/52 (19%) achieved a best response of undetectable MRD in bone marrow (BM, 44 pts tested), and 14/52 (27%) in peripheral blood (PB, 34 pts tested). 4-yr PFS and OS are 74% and 93% (Fig. 1B-C), with 3 deaths (n=1 each: sudden death, MDS, Richter’s syndrome). Pts who achieved BM and PB undetectable MRD had a significantly larger decrease in circulating CCL4 (p=0.02) and CXCL13 levels (p=0.01), respectively, comparing baseline to 1 week of combination tx (Fig. 1D). Image:Summary/Conclusion: Ibr + obin is a highly active combination in R/R CLL, achieving 50% CR and 4-yr PFS/OS of 74%/93%, suggesting a potential positive impact of obin (though possibly also reflecting an earlier relapse population than RESONATE). Responses were observed in all 3 pts who had progressed after venetoclax, including 1 CR. With ~3.5 yrs median follow-up, and some pts on tx over 6 yrs, cardiotoxicity was consistent with prior ibr studies, and no new safety concerns have emerged. Our data support continued exploration of combination BTK inhibitor plus obin in R/R CLL.