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Combination Targeted Therapy in Relapsed Diffuse Large B-Cell Lymphoma. BACKGROUND: The identification of oncogenic mutations in diffuse large B-cell lymphoma (DLBCL) has led to the development of drugs that target essential survival pathways, but whether targeting multiple survival pathways may be curative in DLBCL is unknown. METHODS: We performed a single-center, phase 1b-2 study of a regimen of venetoclax, ibrutinib, prednisone, obinutuzumab, and lenalidomide (ViPOR) in relapsed or refractory DLBCL. In phase 1b, which included patients with DLBCL and indolent lymphomas, four dose levels of venetoclax were evaluated to identify the recommended phase 2 dose, with fixed doses of the other four drugs. A phase 2 expansion in patients with germinal-center B-cell (GCB) and non-GCB DLBCL was performed. ViPOR was administered every 21 days for six cycles. RESULTS: In phase 1b of the study, involving 20 patients (10 with DLBCL), a single dose-limiting toxic effect of grade 3 intracranial hemorrhage occurred, a result that established venetoclax at a dose of 800 mg as the recommended phase 2 dose. Phase 2 included 40 patients with DLBCL. Toxic effects that were observed among all the patients included grade 3 or 4 neutropenia (in 24% of the cycles), thrombocytopenia (in 23%), anemia (in 7%), and febrile neutropenia (in 1%). Objective responses occurred in 54% of 48 evaluable patients with DLBCL, and complete responses occurred in 38%; complete responses were exclusively in patients with non-GCB DLBCL and high-grade B-cell lymphoma with rearrangements of MYC and BCL2 or BCL6 (or both). Circulating tumor DNA was undetectable in 33% of the patients at the end of ViPOR therapy. With a median follow-up of 40 months, 2-year progression-free survival and overall survival were 34% (95% confidence interval [CI], 21 to 47) and 36% (95% CI, 23 to 49), respectively. CONCLUSIONS: Treatment with ViPOR was associated with durable remissions in patients with specific molecular DLBCL subtypes and was associated with mainly reversible adverse events. (Funded by the Intramural Research Program of the National Cancer Institute and the National Center for Advancing Translational Sciences of the National Institutes of Health and others; ClinicalTrials.gov number, NCT03223610.).

Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogenous disease for which chemoimmunotherapy can be curative.1-7 Patients with early relapse or refractory (R/R) disease, however, have a poor outcome with conventional treatments.8,9 While anti-CD19 chimeric antigen receptor T-cell (CAR-T) therapy has improved outcomes, only approximately 30-40% of R/R patients are cured with such therapy.10-13 Genetic and functional genomic studies have revealed oncogenic driver pathways in DLBCL, leading to the development of drugs against actionable targets. Although many of these agents are active as monotherapy,14-17 they rarely induce deep responses or cure, although certain molecular subtypes may occasionally and exceptionally respond to these agents.18-20 Based on our identification of synergy of targeted drugs in DLBCL models,21,22 we hypothesized that inhibiting multiple survival pathways simultaneously could be curative in certain DLBCL molecular subtypes.

es or cure, although certain molecular subtypes may occasionally and exceptionally respond to these agents.18-20 Based on our identification of synergy of targeted drugs in DLBCL models,21,22 we hypothesized that inhibiting multiple survival pathways simultaneously could be curative in certain DLBCL molecular subtypes. Survival pathways in the activated B-cell (ABC) subtype of DLBCL include chronic active B-cell receptor (BCR) signaling to NF-κB and phosphoinositide 3-kinase (PI3K), prevention of apoptosis by BCL2, and expression of the lineage-restricted transcription factors IRF4, Ikaros (IKZF1) and Aiolos (IKZF3).23,24 In the germinal center B-cell (GCB) subtype of DLBCL, cell viability is maintained by constitutive BCR-dependent PI3K signaling, BCL2, Ikaros, and Aiolos.25,26 These survival pathways can be blocked by targeted drugs, including the BTK inhibitor ibrutinib, which targets BCR-dependent NF-κB activation, glucocorticoids, which target proximal BCR signaling,27 the BCL2 inhibitor venetoclax, and lenalidomide, which targets Ikaros, Aiolos and, indirectly, IRF4. Preclinical studies have demonstrated synergistic killing of DLBCL cell lines by combinations of these drugs.21,22

b, which targets BCR-dependent NF-κB activation, glucocorticoids, which target proximal BCR signaling,27 the BCL2 inhibitor venetoclax, and lenalidomide, which targets Ikaros, Aiolos and, indirectly, IRF4. Preclinical studies have demonstrated synergistic killing of DLBCL cell lines by combinations of these drugs.21,22 We developed a 5-drug regimen (ViPOR) that targets these pathways and includes obinutuzumab to trigger innate immune responses to malignant B-cells. (Fig. 1A). To maximize potential synergy while minimizing cumulative drug toxicities, we administered all agents in non-continuous cycles for a fixed duration.21,22 Herein, we present the results of a single-center phase Ib study of ViPOR in R/R B-cell lymphomas and efficacy analysis for the phase II expansion in R/R DLBCL.

R/R B-cell lymphoma patients were eligible for the phase Ib and R/R DLBCL (including high-grade B-cell lymphoma “double-hit” (HGBCL-DH) and T-cell/histocyte-rich large B-cell lymphoma (THRLBCL)) for the phase II expansion (Supplementary Methods). Inclusion criteria included age ≥18 years, ECOG performance status ≤2, and adequate organ function unless secondary to lymphoma. Prior anthracycline was required for DLBCL patients, and all patients required prior anti-CD20 antibody. Exclusions included active CNS disease, pregnant or breastfeeding women, use of strong CYP3A4 inducers/inhibitors, and HIV infection. Prior treatment with one study agent (i.e., venetoclax, ibrutinib, or lenalidomide) was allowed. The study was approved by the National Cancer Institute Institutional Review Board, and all patients provided written informed consent in accordance with the Declaration of Helsinki (protocol available at nejm.org). ClinicalTrials.gov identifier: NCT03223610.

(i.e., venetoclax, ibrutinib, or lenalidomide) was allowed. The study was approved by the National Cancer Institute Institutional Review Board, and all patients provided written informed consent in accordance with the Declaration of Helsinki (protocol available at nejm.org). ClinicalTrials.gov identifier: NCT03223610. In phase Ib, a “3+3” design was used to determine the recommended phase 2 dose (RP2D) of venetoclax at 4 dose-levels (DLs) (200-800 mg) days 2-14 (starting cycle 2 for DL1) (Table S1) in combination with fixed-dose ibrutinib 560 mg days 1-14, prednisone 100 mg days 1-7, obinutuzumab 1000 mg days 1-2, and lenalidomide 15 mg days 1-14 (Fig. 1B). A 12-day initial ramp-up of venetoclax was used in all patients (Table S2) with serial tumor lysis syndrome laboratory monitoring and prophylaxis based on pre-treatment tumor lysis syndrome risk (Supplementary Methods). Dose-limiting toxicity (DLT) criteria are provided in the Supplementary Appendix.

ys 1-14 (Fig. 1B). A 12-day initial ramp-up of venetoclax was used in all patients (Table S2) with serial tumor lysis syndrome laboratory monitoring and prophylaxis based on pre-treatment tumor lysis syndrome risk (Supplementary Methods). Dose-limiting toxicity (DLT) criteria are provided in the Supplementary Appendix. Phase II expansion cohorts, 20 patients each with GCB and non-GCB DLBCL, were assessed at the RP2D. Six cycles of ViPOR every 21-days were administered unless progression or unacceptable toxicity. All patients received growth factor support and pneumocystis jirovecii prophylaxis, with venous thromboembolism prophylaxis given if indicated (Supplementary Methods). Pre-treatment biopsies were analyzed for whole-exome and transcriptome sequencing (Supplementary Methods). Baseline computed tomography (CT), fluorodeoxyglucose positron-emission tomography (FDG-PET), and bone marrow biopsy/aspirate were performed with restaging CT and PET scans during treatment and in follow-up (Fig. 1B and Supplementary Methods). Plasma samples were collected for circulating-tumor DNA (ctDNA) analysis using clonoSEQ (Fig. 1B),28 as well as for pharmacokinetic (PK) analyses (Supplementary Methods). Response to therapy was per Lugano classification criteria.29

CT and PET scans during treatment and in follow-up (Fig. 1B and Supplementary Methods). Plasma samples were collected for circulating-tumor DNA (ctDNA) analysis using clonoSEQ (Fig. 1B),28 as well as for pharmacokinetic (PK) analyses (Supplementary Methods). Response to therapy was per Lugano classification criteria.29 In phase Ib, a maximum of 30 patients could be treated to assess all 4 DLs of venetoclax and allow for a potential DL(−1). In phase II, an additional 40 R/R DLBCL patients were treated at the RP2D. With 40 R/R DLBCL patients, an exact binomial test with a 0.10 one-sided significance level has 87% power to rule out 40% and target a 60% overall response rate. Response duration, progression-free survival, and overall survival were determined using the Kaplan-Meier method, with the median and a 95% confidence interval (95% CI) reported for each analysis, and a data cutoff date of July 11, 2023. Response duration was calculated in all responding patients (N=26) from the date of first response to date of relapse/progression or last follow-up. Progression-free and overall survival were calculated in all DLBCL patients (N=50) from the on-study date to date of relapse/progression, death, or last follow-up, as appropriate. PET and minimal residual disease (MRD) analyses based on data obtained after cycles 1 or 2, or at the end-of-therapy began at that respective timepoint. Additional statistical methods are described in the Supplementary Appendix.

In phase Ib, a “3+3” design was used to determine the recommended phase 2 dose (RP2D) of venetoclax at 4 dose-levels (DLs) (200-800 mg) days 2-14 (starting cycle 2 for DL1) (Table S1) in combination with fixed-dose ibrutinib 560 mg days 1-14, prednisone 100 mg days 1-7, obinutuzumab 1000 mg days 1-2, and lenalidomide 15 mg days 1-14 (Fig. 1B). A 12-day initial ramp-up of venetoclax was used in all patients (Table S2) with serial tumor lysis syndrome laboratory monitoring and prophylaxis based on pre-treatment tumor lysis syndrome risk (Supplementary Methods). Dose-limiting toxicity (DLT) criteria are provided in the Supplementary Appendix. Phase II expansion cohorts, 20 patients each with GCB and non-GCB DLBCL, were assessed at the RP2D. Six cycles of ViPOR every 21-days were administered unless progression or unacceptable toxicity. All patients received growth factor support and pneumocystis jirovecii prophylaxis, with venous thromboembolism prophylaxis given if indicated (Supplementary Methods). Pre-treatment biopsies were analyzed for whole-exome and transcriptome sequencing (Supplementary Methods). Baseline computed tomography (CT), fluorodeoxyglucose positron-emission tomography (FDG-PET), and bone marrow biopsy/aspirate were performed with restaging CT and PET scans during treatment and in follow-up (Fig. 1B and Supplementary Methods). Plasma samples were collected for circulating-tumor DNA (ctDNA) analysis using clonoSEQ (Fig. 1B),28 as well as for pharmacokinetic (PK) analyses (Supplementary Methods). Response to therapy was per Lugano classification criteria.29

In phase Ib, a maximum of 30 patients could be treated to assess all 4 DLs of venetoclax and allow for a potential DL(−1). In phase II, an additional 40 R/R DLBCL patients were treated at the RP2D. With 40 R/R DLBCL patients, an exact binomial test with a 0.10 one-sided significance level has 87% power to rule out 40% and target a 60% overall response rate. Response duration, progression-free survival, and overall survival were determined using the Kaplan-Meier method, with the median and a 95% confidence interval (95% CI) reported for each analysis, and a data cutoff date of July 11, 2023. Response duration was calculated in all responding patients (N=26) from the date of first response to date of relapse/progression or last follow-up. Progression-free and overall survival were calculated in all DLBCL patients (N=50) from the on-study date to date of relapse/progression, death, or last follow-up, as appropriate. PET and minimal residual disease (MRD) analyses based on data obtained after cycles 1 or 2, or at the end-of-therapy began at that respective timepoint. Additional statistical methods are described in the Supplementary Appendix.

Sixty patients with R/R B-cell lymphoma enrolled between February 2018 and June 2021, including 20 patients (10 DLBCL) in phase Ib and 40 patients (all DLBCL) in phase II (Table 1). In 50 DLBCL patients, the median (range) age was 61 (29-77) years, with stage III/IV disease in 92%, elevated LDH in 86%, ≥2 extranodal sites in 56%, and international prognostic index (IPI) ≥3 in 68% of patients. Seventeen (34%) DLBCL patients had transformed lymphoma. Median (range) prior systemic therapies was 3 (1-9), including 20 patients (40%) with prior CAR-T and 29 (58%) with refractory disease per SCHOLAR-1 criteria.9 Twenty-five patients had DLBCL, NOS, including 12 GCB and 13 non-GCB subtypes by immunohistochemistry.30,31 Twenty patients had HGBCL-DH, including 17 with MYC/BCL2 translocations (HGBCL-DH-BCL2) and 3 with MYC/BCL6 translocations (HGBCL-DH-BCL6), and five patients had THRLBCL (Tables 1 and S3).31 Eighteen of 20 patients in phase Ib were evaluable for DLTs (2 patients did not complete the DLT window due to progression). One of 18 evaluable patients experienced a DLT of grade 3 intracranial hemorrhage at DL1 in the setting of concomitant enoxaparin and aspirin. No other DLTs occurred, and venetoclax 800 mg was identified as the RP2D.

ts in phase Ib were evaluable for DLTs (2 patients did not complete the DLT window due to progression). One of 18 evaluable patients experienced a DLT of grade 3 intracranial hemorrhage at DL1 in the setting of concomitant enoxaparin and aspirin. No other DLTs occurred, and venetoclax 800 mg was identified as the RP2D. Hematologic adverse events of any grade occurred in over two-thirds of patients (Fig. 2), with grade 3-4 neutropenia, thrombocytopenia, and anemia observed in 24%, 23%, and 7% of cycles, respectively (Tables S4-S5). Furthermore, only 3 episodes of grade 3 febrile neutropenia occurred, and T-cell lymphocyte subsets were generally unaffected following therapy (Fig. S1, Table S6, and Supplementary Results). Although hypokalemia was the only non-hematologic grade 3-4 adverse event observed in ≥10% of patients (28%), 67% of patients experienced hypokalemia of any grade which required electrolyte replacement. Other non-hematologic adverse events included diarrhea (68%), nausea (45%), rash (35%), and fatigue (33%) (Fig. 2). Grade 3 atrial fibrillation occurred in 3 patients, and 1 major hemorrhage event occurred. One grade 4 tumor lysis syndrome event occurred, resolved, and did not recur despite continued treatment. Serious adverse events occurred in 42% of patients (Tables S7-S8), with non-neutropenic fever most common.

(33%) (Fig. 2). Grade 3 atrial fibrillation occurred in 3 patients, and 1 major hemorrhage event occurred. One grade 4 tumor lysis syndrome event occurred, resolved, and did not recur despite continued treatment. Serious adverse events occurred in 42% of patients (Tables S7-S8), with non-neutropenic fever most common. Dose reductions and delays for toxicity occurred in 17% and 25% of patients, respectively, and 5 patients prematurely discontinued study therapy due to an unacceptable adverse event or intercurrent illness (Supplementary Results). PK analysis of venetoclax, ibrutinib, and lenalidomide was comparable to published single-agent data, indicating no significant drug interactions (Fig. S2, Table S9, and Supplementary Results).32-34 Forty-eight DLBCL patients were evaluable for response (2 HGBCL-DH-BCL2 patients discontinued therapy for toxicity prior to restaging). Response rate was 54% (26/48) with 38% (18/48) complete response (Fig. 1C and Table 2). Median (range) time to response was 0.66 (0.59-4.34) months, with 72% of complete responses ongoing without consolidation (Supplementary Results). Complete responses were obtained in 62% (8/13) of non-GCB DLBCL, NOS, 53% (8/15) of HGBCL-DH-BCL2, 33% (1/3) of HGBCL-DH-BCL6, and 20% (1/5) of THRLBCL, while no complete responses (0/12) were observed in GCB DLBCL, NOS (33% partial response, 4/12) (Table 2).

onses ongoing without consolidation (Supplementary Results). Complete responses were obtained in 62% (8/13) of non-GCB DLBCL, NOS, 53% (8/15) of HGBCL-DH-BCL2, 33% (1/3) of HGBCL-DH-BCL6, and 20% (1/5) of THRLBCL, while no complete responses (0/12) were observed in GCB DLBCL, NOS (33% partial response, 4/12) (Table 2). With a median follow-up of 40 months (interquartile range: 32-51), 2-year progression-free survival was 34% (95% CI: 21-47%) in all DLBCL patients (Fig. 1D). In responding patients, 2-year response duration was 65% overall, including 78% of patients with complete responses and 38% of patients with partial responses to ViPOR (Fig. S3 and 1E). By subtype, 2-year progression-free survival was 39% in non-GCB DLBCL, NOS, 47% in HGBCL-DH-BCL2, 33% in HGBCL-DH-BCL6, 40% in THRLBCL, and 8% in GCB DLBCL, NOS (Fig. 1F). Overall survival largely reflected progression-free survival both overall and within DLBCL subtypes (Fig. S4). DLBCL patients who received ViPOR as second-line therapy had improved progression-free survival compared to those who received it as third or later-line therapy [log-rank hazard ratio (HR) 0.33 (95% CI: 0.17-0.66)] (Table 2 and Fig. S6). Four patients experienced systemic disease relapse after achieving complete response, all within 18 months of treatment, and one patient died in remission 31 months after treatment from COVID-19 infection (Fig. S7). Response and survival data for additional DLBCL subsets, including transformed, post-CAR-T, refractory, and molecular DLBCL subtypes are shown in Table 2 and in the Supplementary Appendix (Fig. S5 and Supplementary Results).

and one patient died in remission 31 months after treatment from COVID-19 infection (Fig. S7). Response and survival data for additional DLBCL subsets, including transformed, post-CAR-T, refractory, and molecular DLBCL subtypes are shown in Table 2 and in the Supplementary Appendix (Fig. S5 and Supplementary Results). Baseline PET imaging was available in all 50 DLBCL patients with 41 having a paired end-of-therapy scan (Table S10 and Supplementary Results). Patients with a Deauville score of 5 at end-of-therapy had inferior progression-free survival [log-rank HR 0.22 (95% CI: 0.10-0.48)] and overall survival [log-rank HR 0.19 (95% CI: 0.08-0.43)] compared to those with a Deauville score of 1-4 (Supplementary Results). Additionally, patients with elevated total metabolic tumor volume (TMTV) or total lesion glycolysis (TLG) above the median at baseline were less likely to achieve complete response and had inferior progression-free and overall survival compared to those below the baseline median (Fig. S8-S9).

4 (Supplementary Results). Additionally, patients with elevated total metabolic tumor volume (TMTV) or total lesion glycolysis (TLG) above the median at baseline were less likely to achieve complete response and had inferior progression-free and overall survival compared to those below the baseline median (Fig. S8-S9). Of 50 DLBCL patients, 90% (45/50) had detectable ctDNA at baseline for MRD analysis (Table S11 and Supplementary Results). Following ViPOR, ctDNA concentration decreased rapidly with 33% of all patients and 93% of patients in complete response by PET having undetectable ctDNA at the end-of-therapy (Fig. S10). Elevated baseline ctDNA concentration ≥2.5 log10 hGE/mL was associated with inferior progression-free and overall survival to ViPOR, whereas undetectable ctDNA after cycle 1, cycle 2, or at end-of-therapy was associated with improved progression-free and overall survival compared to those with detectable ctDNA (Fig. S11-S12). Of 15 patients in complete response with serial ctDNA samples, 11 remained progression-free with undetectable ctDNA, whereas among 4 patients who relapsed, 3 developed molecular relapse before clinical relapse appeared by imaging (Fig. S13).

e and overall survival compared to those with detectable ctDNA (Fig. S11-S12). Of 15 patients in complete response with serial ctDNA samples, 11 remained progression-free with undetectable ctDNA, whereas among 4 patients who relapsed, 3 developed molecular relapse before clinical relapse appeared by imaging (Fig. S13). To explore the basis for ViPOR sensitivity, we employed high-throughput drug screens to measure the cytotoxicity of the 4 small molecule ViPOR drugs in pair-wise dose-response titrations in ABC cell line models (N=4), and used the ΔBliss metric to quantify drug synergy (Fig. 3A).21 Interestingly, while synergistic cytotoxicity was observed with all drug doublets, the pattern of synergy varied across ABC models. For example, the combination of prednisolone, the active metabolite of prednisone, with venetoclax was synergistically toxic in two of the four ABC models, while prednisolone plus lenalidomide was synergistic in a different pair of models, and prednisolone plus ibrutinib was synergistic in all four models (Fig. 3B). Furthermore, this was in contrast to the lack of synergistic cytotoxicity observed in most GCB cell line models (Fig. S14). Using a quantitative measure of apoptosis, we tested each ViPOR drug individually and as a 4-drug combination over a 2-to-24-hour time course. In two ABC models, the 4-drug combination produced more rapid and profound apoptosis than any individual ViPOR agent (Fig. 3C). Thus, while individual DLBCL models respond differentially to ViPOR drug doublets, the combination of all four ViPOR drugs overcame this functional heterogeneity.

r a 2-to-24-hour time course. In two ABC models, the 4-drug combination produced more rapid and profound apoptosis than any individual ViPOR agent (Fig. 3C). Thus, while individual DLBCL models respond differentially to ViPOR drug doublets, the combination of all four ViPOR drugs overcame this functional heterogeneity. We analyzed whole-exome and RNA-sequencing from ViPOR tumor samples to assign tumors to DLBCL genetic subtypes using the LymphGen algorithm3 and to cell-of-origin gene expression subtypes (Fig. S15-S16). Patients with the MCD subtype (N=6) had a considerably higher complete response rate [5/6, 83% (95% CI: 53-100%)] compared to patients with all other genetically classified DLBCL subtypes [4/18, 22% (95% CI: 3-41%)] and in all non-MCD patients [12/38; 32% (95% CI: 22-42%)] (Fig. 3D). Additionally, the 1 patient with N1 DLBCL also achieved a complete response. Among the gene-expression subtypes, complete response rate was highest in ABC (8/14, 57%) and unclassified DLBCL (3/8, 38%), and lowest in GCB DLBCL (6/22, 27%). Together, these data support the hypothesis that the MCD and possibly N1 genetic subtypes of ABC DLBCL likely benefitted from ViPOR due to their exceptional reliance on BCR-dependent NF-κB activation.18,19

sponse rate was highest in ABC (8/14, 57%) and unclassified DLBCL (3/8, 38%), and lowest in GCB DLBCL (6/22, 27%). Together, these data support the hypothesis that the MCD and possibly N1 genetic subtypes of ABC DLBCL likely benefitted from ViPOR due to their exceptional reliance on BCR-dependent NF-κB activation.18,19 Patients with HGBCL-DH-BCL2 had a high rate of durable complete response following ViPOR. Such tumors are classified as EZB-MYC+ in the DLBCL genetic taxonomy, whereas other GCB DLBCLs are classified into the EZB-MYC−, ST2, and BN2 subtypes (Fig. 3E).3 The complete response rate was higher in EZB-MYC+ (3/6, 50%), than in EZB-MYC− (0/6, 0%) with ViPOR, suggesting the dual translocations of MYC and BCL2 might confer sensitivity to ViPOR. To test this hypothesis, we measured the cytotoxicity of the 4 VIPOR agents in 12 GCB DLBCL cell line models, including EZB-MYC+(N=5), EZB-MYC− (N=4), ST2 (N=2), and genetically unclassified GCB (N=1) models. EZB-MYC+ lines were strikingly more sensitive to venetoclax than EZB-MYC− or other GCB models, whereas these lines did not differ consistently to the other ViPOR agents (Fig. 3E). This finding suggests that BCL2 inhibition with venetoclax likely contributes prominently to the observed durable complete response rate to ViPOR in patients with EZB-MYC+DLBCL.

Sixty patients with R/R B-cell lymphoma enrolled between February 2018 and June 2021, including 20 patients (10 DLBCL) in phase Ib and 40 patients (all DLBCL) in phase II (Table 1). In 50 DLBCL patients, the median (range) age was 61 (29-77) years, with stage III/IV disease in 92%, elevated LDH in 86%, ≥2 extranodal sites in 56%, and international prognostic index (IPI) ≥3 in 68% of patients. Seventeen (34%) DLBCL patients had transformed lymphoma. Median (range) prior systemic therapies was 3 (1-9), including 20 patients (40%) with prior CAR-T and 29 (58%) with refractory disease per SCHOLAR-1 criteria.9 Twenty-five patients had DLBCL, NOS, including 12 GCB and 13 non-GCB subtypes by immunohistochemistry.30,31 Twenty patients had HGBCL-DH, including 17 with MYC/BCL2 translocations (HGBCL-DH-BCL2) and 3 with MYC/BCL6 translocations (HGBCL-DH-BCL6), and five patients had THRLBCL (Tables 1 and S3).31

Eighteen of 20 patients in phase Ib were evaluable for DLTs (2 patients did not complete the DLT window due to progression). One of 18 evaluable patients experienced a DLT of grade 3 intracranial hemorrhage at DL1 in the setting of concomitant enoxaparin and aspirin. No other DLTs occurred, and venetoclax 800 mg was identified as the RP2D. Hematologic adverse events of any grade occurred in over two-thirds of patients (Fig. 2), with grade 3-4 neutropenia, thrombocytopenia, and anemia observed in 24%, 23%, and 7% of cycles, respectively (Tables S4-S5). Furthermore, only 3 episodes of grade 3 febrile neutropenia occurred, and T-cell lymphocyte subsets were generally unaffected following therapy (Fig. S1, Table S6, and Supplementary Results). Although hypokalemia was the only non-hematologic grade 3-4 adverse event observed in ≥10% of patients (28%), 67% of patients experienced hypokalemia of any grade which required electrolyte replacement. Other non-hematologic adverse events included diarrhea (68%), nausea (45%), rash (35%), and fatigue (33%) (Fig. 2). Grade 3 atrial fibrillation occurred in 3 patients, and 1 major hemorrhage event occurred. One grade 4 tumor lysis syndrome event occurred, resolved, and did not recur despite continued treatment. Serious adverse events occurred in 42% of patients (Tables S7-S8), with non-neutropenic fever most common.

(33%) (Fig. 2). Grade 3 atrial fibrillation occurred in 3 patients, and 1 major hemorrhage event occurred. One grade 4 tumor lysis syndrome event occurred, resolved, and did not recur despite continued treatment. Serious adverse events occurred in 42% of patients (Tables S7-S8), with non-neutropenic fever most common. Dose reductions and delays for toxicity occurred in 17% and 25% of patients, respectively, and 5 patients prematurely discontinued study therapy due to an unacceptable adverse event or intercurrent illness (Supplementary Results). PK analysis of venetoclax, ibrutinib, and lenalidomide was comparable to published single-agent data, indicating no significant drug interactions (Fig. S2, Table S9, and Supplementary Results).32-34

Forty-eight DLBCL patients were evaluable for response (2 HGBCL-DH-BCL2 patients discontinued therapy for toxicity prior to restaging). Response rate was 54% (26/48) with 38% (18/48) complete response (Fig. 1C and Table 2). Median (range) time to response was 0.66 (0.59-4.34) months, with 72% of complete responses ongoing without consolidation (Supplementary Results). Complete responses were obtained in 62% (8/13) of non-GCB DLBCL, NOS, 53% (8/15) of HGBCL-DH-BCL2, 33% (1/3) of HGBCL-DH-BCL6, and 20% (1/5) of THRLBCL, while no complete responses (0/12) were observed in GCB DLBCL, NOS (33% partial response, 4/12) (Table 2).

Baseline PET imaging was available in all 50 DLBCL patients with 41 having a paired end-of-therapy scan (Table S10 and Supplementary Results). Patients with a Deauville score of 5 at end-of-therapy had inferior progression-free survival [log-rank HR 0.22 (95% CI: 0.10-0.48)] and overall survival [log-rank HR 0.19 (95% CI: 0.08-0.43)] compared to those with a Deauville score of 1-4 (Supplementary Results). Additionally, patients with elevated total metabolic tumor volume (TMTV) or total lesion glycolysis (TLG) above the median at baseline were less likely to achieve complete response and had inferior progression-free and overall survival compared to those below the baseline median (Fig. S8-S9). Of 50 DLBCL patients, 90% (45/50) had detectable ctDNA at baseline for MRD analysis (Table S11 and Supplementary Results). Following ViPOR, ctDNA concentration decreased rapidly with 33% of all patients and 93% of patients in complete response by PET having undetectable ctDNA at the end-of-therapy (Fig. S10). Elevated baseline ctDNA concentration ≥2.5 log10 hGE/mL was associated with inferior progression-free and overall survival to ViPOR, whereas undetectable ctDNA after cycle 1, cycle 2, or at end-of-therapy was associated with improved progression-free and overall survival compared to those with detectable ctDNA (Fig. S11-S12). Of 15 patients in complete response with serial ctDNA samples, 11 remained progression-free with undetectable ctDNA, whereas among 4 patients who relapsed, 3 developed molecular relapse before clinical relapse appeared by imaging (Fig. S13).

To explore the basis for ViPOR sensitivity, we employed high-throughput drug screens to measure the cytotoxicity of the 4 small molecule ViPOR drugs in pair-wise dose-response titrations in ABC cell line models (N=4), and used the ΔBliss metric to quantify drug synergy (Fig. 3A).21 Interestingly, while synergistic cytotoxicity was observed with all drug doublets, the pattern of synergy varied across ABC models. For example, the combination of prednisolone, the active metabolite of prednisone, with venetoclax was synergistically toxic in two of the four ABC models, while prednisolone plus lenalidomide was synergistic in a different pair of models, and prednisolone plus ibrutinib was synergistic in all four models (Fig. 3B). Furthermore, this was in contrast to the lack of synergistic cytotoxicity observed in most GCB cell line models (Fig. S14). Using a quantitative measure of apoptosis, we tested each ViPOR drug individually and as a 4-drug combination over a 2-to-24-hour time course. In two ABC models, the 4-drug combination produced more rapid and profound apoptosis than any individual ViPOR agent (Fig. 3C). Thus, while individual DLBCL models respond differentially to ViPOR drug doublets, the combination of all four ViPOR drugs overcame this functional heterogeneity.

We provide evidence that the simultaneous targeting of multiple survival pathways is potentially curative in specific molecular subtypes of DLBCL. To optimize multi-agent drug synergy, we designed a schedule reminiscent of combination chemotherapy, whereby all targeted agents are concurrently administered in a non-continuous fashion for a maximum of 6 cycles. This strategy avoids the cumulative toxicity associated with continuous and/or indefinite drug dosing, thereby allowing the safe administration of multiple targeted agents. Although hematologic toxicities were common and occurred in over two-thirds of patients, serious hematologic adverse events occurred in less than a quarter of cycles with febrile neutropenia observed in only 1% of cycles. Intermittent drug dosing also resulted in less non-hematologic toxicities, such as high-grade rash, compared to other continuous targeted-therapy regimens.35-37 Despite an initial concern for possible tumor lysis syndrome and hyperkalemia, diarrhea and hypokalemia were more commonly observed following ViPOR, with most patients requiring anti-diarrheal and electrolyte support in contrast to the 1 tumor lysis syndrome event observed. Furthermore, adverse events frequently resolved during the off-treatment week and most patients received all cycles on schedule and without dose reductions.

re commonly observed following ViPOR, with most patients requiring anti-diarrheal and electrolyte support in contrast to the 1 tumor lysis syndrome event observed. Furthermore, adverse events frequently resolved during the off-treatment week and most patients received all cycles on schedule and without dose reductions. Due to the molecular heterogeneity and complex aberrant genetic landscape of DLBCL, we hypothesized that the inhibition of multiple driver pathways is necessary for the curative potential of targeted therapy. ViPOR synergistically targets key survival pathways in non-GCB DLBCL, including BCR-dependent NF-κB signaling using the combination of ibrutinib, lenalidomide, and prednisone, 21,22,23,27 as well as the anti-apoptotic protein BCL2 with venetoclax. In line with this hypothesis, ViPOR achieved a higher complete response rate (62%) and 2-year progression-free survival (39%) in non-GCB DLBCL than in GCB DLBCL, NOS. Notably, MCD DLBCL had a higher complete response rate (83%) than other genetic DLBCL subtypes, which is in line with previous laboratory and clinical evidence of exceptional dependence of MCD tumors on BCR signaling.15,19,20,24,38

62%) and 2-year progression-free survival (39%) in non-GCB DLBCL than in GCB DLBCL, NOS. Notably, MCD DLBCL had a higher complete response rate (83%) than other genetic DLBCL subtypes, which is in line with previous laboratory and clinical evidence of exceptional dependence of MCD tumors on BCR signaling.15,19,20,24,38 Unexpectedly, ViPOR was highly active in GCB tumors with MYC and BCL2 translocations (HGBCL-DH-BCL2), with a higher 2-year progression-free survival (47%) than in GCB tumors lacking both translocations (8%). Mechanistically, MYC can sensitize cells to apoptosis, but BCL2 can block this toxicity.39 Venetoclax was more toxic for cell line models of HGBCL-DH-BCL2 than for other GCB DLBCL lines, leading us to speculate that the inclusion of venetoclax in ViPOR may expose HGBCL-DH-BCL2 tumors to the toxicity of MYC. Among post-CAR-T patients, ViPOR achieved a 2-year progression-free survival of 30%, suggesting it may alter the poor prognosis of these patients.40 Additionally, ViPOR allowed successful bridging to radiotherapy, CAR-T, or allogeneic transplant in over one-third of patients who achieved partial response. With a median follow-up of 40 months, over one-third of all DLBCL patients were progression-free at 2-years, with 78% of complete responses durable without consolidation. Not unexpectedly, ViPOR yielded a higher complete response rate and progression-free survival when administered as second-line therapy, suggesting it should be considered early in R/R DLBCL.

r one-third of all DLBCL patients were progression-free at 2-years, with 78% of complete responses durable without consolidation. Not unexpectedly, ViPOR yielded a higher complete response rate and progression-free survival when administered as second-line therapy, suggesting it should be considered early in R/R DLBCL. PET and molecular biomarkers of disease were prognostic of outcome following ViPOR. Elevated baseline metabolic (i.e., TMTV and TLG) or molecular (i.e., ctDNA) disease burden was associated with inferior progression-free and overall survival. During therapy, ctDNA provided an early predictor of long-term outcome as did undetectable ctDNA at the end-of-therapy.

re prognostic of outcome following ViPOR. Elevated baseline metabolic (i.e., TMTV and TLG) or molecular (i.e., ctDNA) disease burden was associated with inferior progression-free and overall survival. During therapy, ctDNA provided an early predictor of long-term outcome as did undetectable ctDNA at the end-of-therapy. In conclusion, this study shows that multiple targeted agents can be administered concurrently in a safe and tolerable fashion across patients of all ages with R/R DLBCL. Our study also shows that simultaneous targeting of multiple survival pathways is potentially curative in specific molecular subtypes of R/R DLBCL. ViPOR was not curative in GCB DLBCL, NOS, highlighting the need to develop and test agents targeting GCB-specific pathways. Notably, durable complete responses were observed in patients who were heavily pretreated with chemotherapy or CAR-T therapy. As with any phase II study, one must recognize the limitations of sample size and patient selection. While the efficacy of ViPOR in specific molecular subtypes limits the subset of DLBCL patients with potentially curable disease, this very specificity provides some confidence in the generalizability of our results. A multicenter phase II study is under development to confirm the activity of ViPOR in patients with R/R non-GCB DLBCL and HGBCL-DH-BCL2.