Chronic Active B-cell Receptor Signaling Research Articles

Antigen-independent, autonomous B cell receptor signaling drives activated B cell DLBCL.

Diffuse large B cell lymphoma of activated B cell type (ABC-DLBCL), a major cell-of-origin DLBCL subtype, is characterized by chronic active B cell receptor (BCR) signaling and NF-κB activation, which can be explained by activating mutations of the BCR signaling cascade in a minority of cases. We demonstrate that autonomous BCR signaling, akin to its essential pathogenetic role in chronic lymphocytic leukemia (CLL), can explain chronic active BCR signaling in ABC-DLBCL. 13 of 18 tested DLBCL-derived BCR, including 12 cases selected for expression of IgM, induced spontaneous calcium flux and increased phosphorylation of the BCR signaling cascade in murine triple knockout pre-B cells without antigenic stimulation or external BCR crosslinking. Autonomous BCR signaling was associated with IgM isotype, dependent on somatic BCR mutations and individual HCDR3 sequences, and largely restricted to non-GCB DLBCL. Autonomous BCR signaling represents a novel immunological oncogenic driver mechanism in DLBCL originating from individual BCR sequences and adds a new dimension to currently proposed genetics- and transcriptomics-based DLBCL classifications.

Optogenetic Control of Oncogenic Signaling in B-Cell Malignancies

Background: B-cell receptor (BCR) signals are essential determinants of survival and proliferation throughout normal B-cell development. In B-cell malignancies, these signals are frequently generated by oncogenic mimics of the BCR signaling pathway. For instance, oncogenes in B-ALL, derived from B-cell precursors, typically mimic survival signals from a constitutively active pre-BCR, while tonic and chronic active BCR signaling were identified in mature B-cell lymphomas. Oncogenic BCR-signaling represents an important target of therapeutic intervention: for instance, small molecule inhibitors of SYK (e.g. entospletinib) and BTK (e.g. ibrutinib) have been developed to disrupt oncogenic BCR signaling in mature B-cell lymphomas. Besides SYK and BTK tyrosine kinases, oncogenic BCR-signaling leads to activation of PLCG2, which initiates the release of Ca 2+ from the ER into the cytoplasm. Thereby, calcium flux is decoded by NFATC1 or NF-kB based on fast (NFATC1) or slow (NF-kB) frequencies of Ca 2+-signals. Significance: To interrogate Ca 2+-signals as critical integration point of oncogenic BCR-signaling, we engineered cell lines and PDX with a GCaMP6s-biosensor, which allows tracing of calcium signaling in single cells over time ( Figure A). To cover B-cell malignancies from multiple stages of B-cell development, we engineered B-ALL (pro- and pre-B cells), mantle cell lymphoma (MCL, naïve B-cells), Burkitt's lymphoma (germinal center), DLBCL (post-GC), multiple myeloma (terminally differentiated plasma cells) and Hodgkin's disease (“crippled” BCR-deficient GC-B cells). Strikingly, B-cell malignancies exhibit autonomous Ca 2+-oscillations, which decreased in their frequency from 20 mHz (pro- and pre-B), 11 mHz (naïve), 4 mHz (germinal center) to 0 mHz in post-GC and terminally differentiated plasma cells based on the differentiation stage of their cell of origin ( Figure A). Results: Given the striking differences in oncogenic BCR-signaling as measured by autonomous Ca 2+ oscillations, we developed an optogenetic system to control the frequency and amplitude of Ca 2+ oscillations by blue light pulses ( Figure B). To model frequency-modulated Ca 2+ signaling in B-cells, we engineered murine pre-B cells carrying an optogenetic tool termed OptoCRAC, which enables reversible activation and inactivation of the plasma membrane Ca 2+ channel Orai1 by intermittent blue light irradiation. Time-lapse imaging of a fluorescent Ca 2+ reporter (R-CaMP1.07) confirmed rapid and reversible Ca 2+ influx in response to intermittent blue light pulses. To assess the phenotypic consequence of high-frequency Ca 2+ oscillations, we developed an LED-array-based optogenetic platform for cell culture plates. While low-frequency Ca 2+ oscillations (0.5 mHz) did not impact cell viability, high-frequency Ca 2+ oscillations (20 mHz) rapidly induced NFAT activation (5 min), and eventually cell death within 72 h ( Figure B). Mechanism and conclusions: To elucidate the mechanistic contribution of NFATC1 in B-cell death induced by fast Ca 2+ oscillations, we tested whether genetic deletion of Nfatc1 is sufficient to rescue B-cell death upon fast Ca 2+ oscillation induced by 20 mHz intermittent blue light pulses. As expected, while B-ALL cells retaining intact Nfatc1 rapidly decreased cell viability, the cells with Cre-mediated deletion persistently remained in cell culture upon 20 mHz Ca 2+ oscillation. In addition to genetic deletion of Nfatc1, we also found that inducible expression of mutant activators of NF-kB signaling were able to rescue B-cells from cell death induced by high-frequency oscillations: Concurrent expression of Card11 L232LI, MYD88 L265P and IKK2 S177E/S181E not only reduced the frequency of autonomous Ca 2+ oscillations but also enabled B-cell survival despite delivery of light-pulses and Ca 2+ oscillations at a fast 20 mHz frequency. We conclude that autonomous Ca 2+ oscillations represent a critical integration point of oncogenic BCR-signaling in B-cell malignancies and that optogenetic control of BCR-downstream Ca 2+ oscillations will reveal previously unrecognized vulnerabilities that can be exploited to increase efficacy of BCR-signaling inhibitors (e.g. ibrutinib, entospletinib, idelalisib) that are currently being used for the treatment of B-cell lymphomas.

High-Dose Methotrexate, Ibrutinib and Temozolomide (MIT) for the Treatment of Newly Diagnosed Primary Central Nervous System Lymphoma: A Multi-Center Prospective Phase II Study

Abstract Background: Primary central nervous system lymphoma (PCNSL) is a rare and aggressive subtype of B-cell lymphoma with poor survival. Genetic alterations often occur in the chronic active B-cell receptor signaling which mediate the response to BTK inhibition (BTKi) in PCNSL. However, the efficacy of traditional high-dose methotrexate-based regimen as a first-line treatment was suboptimal with a short effective remission time and low response rate. Therefore, we initiated this phase II trial to investigate the efficacy of novel induction combination of high-dose methotrexate (HD-MTX) plus ibrutinib and temozolomide (MIT), in the treatment of newly diagnosed PCNSL for the first time (ClinicalTrials.gov identifier: NCT04514393). Methods: Patients aged 18-75 years, newly diagnosed PCNSL and at least one measurable or evaluable lesion were enrolled. The MIT induction treatment included six cycles of HD-MTX (3.5 g/m 2, every three weeks), ibrutinib (560 mg/d, after HD-MTX clearance), and temozolomide (150 mg/m2 d1-d5, every three weeks). After the completion of the six cycles of MIT induction treatment, ASCT consolidation (only for patients < 65 years) or daily ibrutinib maintenance (560 mg/d) were administered up to two years, or until disease progression, intolerable toxicity, or death. Treatment response was evaluated by brain MRI and FDG-PET and/or cerebrospinal fluid (CSF) examination every two cycles of MIT treatment. The primary objective of this study was to evaluate the overall response rate (ORR) of MIT induction treatment. The safety profile of MIT was also investigated. At the same time, next generation sequencing (NGS) of baseline tissues and dynamic monitoring with CSF/ plasma ctDNA were performed by Nanjing Geneseeq Technology Inc. Results: Between July 2021 and October 2022, thirty-three patients were enrolled in this open-label, multi-center phase II prospective clinical study. The median age was 56 years (range: 27-75) and 17 of them were male. Eye involvement occurred in 5 patients (15%). Twenty-five patients (76%) had multiple cerebral lesions. The best ORR was 94% and complete response rate was 73%. The median follow-up was 17.5 months (range: 5.8-21.3 months) for survivors. Twenty-three patients underwent ibrutinib maintenance after 6 cycles of induction therapy. The swimmer plot of the 33 patients was shown in Figure 1. The 1-year progression-free survival was 66.4% (95%CI: 58.1-74.7%) and overall survival was 90.5% (95%CI: 85.2-95.8%) (Figure 2). Treatment-related toxicities were acceptable, with grade 3-4 neutropenia occurring in 9%, thrombocytopenia in 6%, subdural hematoma (SH) in 6%, and atrial fibrillation in 3%. The 2 patients with SH recovered following discontinuation of ibrutinib. No fungal infections and treatment-related deaths were observed. NGS of Tissue/CSF was tested in 30 patients, 19 (63%) of whom were classified as MCD subtype, 2 (7%) as BN2 subtype. The mutational profiles of baseline tissue and/or CSF samples revealed that PIM1 and MYD88 mutations were present in 75.9% and 65.5% of the entire cohort followed by BTG2 (55.2%) and CD79B (51.7%). Patients with dynamic CSF ctDNA monitoring during MIT treatment were analyzed (Figure 1). The consistence of ctDNA clearance in CSF/plasma samples and imaging complete remission was observed. Patients with clearance of ctDNA in the CSF after 2 cycles of MIT treatment achieved better PFS. Conclusions: The novel induction treatment of MIT achieved encouraging responses in newly diagnosed PCNSL patients with acceptable toxicities. The early clearance of ctDNA in CSF may be related to favorable survival. Disclosures No relevant conflicts of interest to declare.

A Randomized, Open-Label, Phase 3 Study of Acalabrutinib in Combination with Rituximab and Reduced Dose CHOP (R-miniCHOP) in Older Adults with Untreated Diffuse Large B-Cell Lymphoma (ARCHED / GLA 2022-1)

Background Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype, and its incidence increases with age. Approximately 40% of patients with DLBCL are older than 70 years. Since the addition of the anti-CD20 antibody rituximab to CHOP regime (cyclophosphamide, doxorubicin, vincristine, and prednisone) prognosis in elderly patients has improved but still remains worse compared to their younger counterparts. Adequate dosage of chemotherapy in older adults is limited by comorbidities/frailty, especially in patients >80 years of age. An attenuated chemotherapy regimen, R-miniCHOP, has been evaluated in prospective trials and seems to provide a good balance between safety and efficacy in this vulnerable patient population (Peyrade et al, 2011). However, even with this regime, outcomes remain modest with 2-year overall survival at 50-60%, underlining the need for more effective treatment in this population. Acalabrutinib is a selective, irreversible, second-generation small molecule inhibitor of Bruton Tyrosine Kinase (BTK) with a more favorable safety profile compared to first generation BTK inhibitors. Although, not yet approved for the treatment of DLBCL, acalabrutinib showed promising results both as a single agent (Strati et al, Haematologica 2021) and in combination with full dose R-CHOP (Davies et al, ASH 2022), in patients aged up to 80 years. Furthermore, acalabrutinib carries the potential of improved efficacy in specific subgroups. The activated B-cell like (ABC) DLBCL subtype has been shown to depend on chronic active B-cell receptor signaling involving many downstream kinases including BTK. Additionally, the genetically defined subgroups MCD/C5, N1, and BN2/C1 and also may serve as potential predictive biomarkers for acalabrutinib. Study design and endpoints ARCHED is an investigator initiated, randomized, multicenter, open-label, phase III trial of R-miniCHOP with or without acalabrutinib in older adults with untreated DLBCL. Patients in the standard arm will receive 6 cycles of R-miniCHOP every 21 days followed by 2 cycles of rituximab alone, while patients in the experimental arm will receive the same regimen and additionally acalabrutinib 100 mg p.o. twice daily from day 1 to day 21 for 8 cycles (Figure 1). The primary endpoint of the study is investigator assessed progression-free survival (PFS). Secondary endpoints include overall survival, PFS per blinded independent review, event-free survival, outcomes according to cell of origin and molecular genotype, response rates, duration of response, progression, relapse and central nervous system (CNS) relapse rates as well as toxicity. Key eligibility criteria Patients are eligible for enrolment if they are >80 years old or >60 years old and ineligible for full-dose R-CHOP according to investigator assessment after standardized simplified geriatric evaluation and are diagnosed with untreated CD20+ DLBCL according to the 2017 WHO classification. Primary mediastinal lymphoma, high grade B-cell lymphoma and follicular lymphoma 3B as well as concurrent diagnosis of indolent lymphoma is allowed, but Richter's transformation is an exclusion criterion. Ann Arbor stage I patients can be included only in the presence of concomitant bulky disease (≥ 7.5 cm). Further exclusion criteria are CNS involvement, stroke or intracranial hemorrhage in the past 6 months and relevant organ dysfunction. An ECOG performance status score of 0 - 2 is required for enrolment but an ECOG score of 3 is acceptable if lymphoma associated. Statistical analysis With an expected 5% drop out rate, we aim to recruit 330 patients (165 in each arm) to achieve 80 % power at a 5 % significance level (two-sided) to observe a PFS difference of 15 % in favor of the experimental arm. An interim analysis for efficacy comparing PFS between the two treatment arms will be performed with n=200 patients and 1 year follow-up for the last patient, with approximately 50% of events. Additionally, an interim analysis for safety regarding treatment completion rates and all-cause mortality will take place when 92nd patient in each group has completed the sixth R-mini-CHOP cycle or died. ARCHED is an academic study of the German Lymphoma Alliance and is coordinated by the Saarland University Clinical Trials Unit. This study is funded by AstraZeneca GmbH. The trial is approved in the EU and is open for recruitment in Germany. (EU-CTN: 2022-501187-18-00, NCT05820841).

Phase 2 Response-Adapted Study of Ibrutinib with Temozolomide, Etoposide, Liposomal Doxorubicin, Dexamethasone, and Rituximab (TEDDI-R) for Secondary CNS Lymphoma

Background: Secondary CNS lymphomas (SCNSL) are aggressive and there are few treatment options. CNSLs often have underlying chronic active B-cell receptor signaling that responds to BTK inhibitors, but the response duration is short. TEDDI-R achieves durable remissions in primary DLBCL of the CNS (PCNSL), but the molecular profile of SCNSL tumors that are BTK-responsive is unknown. We present results from an ongoing response-adapted study of ibrutinib with TEDD-R in SCNSL [NCT03964090]. Methods: Pts with aggressive B-cell lymphomas with CNS +/- systemic involvement are eligible if age ≥18, PS<3, and adequate organ function. Pts are untreated with CNS involved or relapsed in the CNS after therapy. Prior BTKi is allowed. HIVis excluded. Baseline tests: brain MRI, PET/CT of body, bone marrow (BM), CSF flow cytometry, and fundoscopic exam. Pts receive isavuconazole as fungal prophylaxis throughout therapy. Pts first receive ibrutinib 560mg x 14d in a treatment window to determine ibrutinib-responsiveness. Pts with ≥20% reduction after ibrutinib receive TEDDI-R, and if <20% reduction receive TEDD-R (no ibrutinib). Chemotherapy is 4 cycles x21d as an outpatient and includes intrathecal chemotherapy. There is no maintenance therapy or planned consolidation. Response is assessed after cycles 2 and 4. Complete responses (CR) are confirmed with FDG-PET brain/body, CSF analysis, and BM. Surveillance scans are q3m for 1y, q4m x 1y, q6m x 1y, then annually. The primary endpoint is PFS. Results:50 pts enrolled between June 2019 and March 2023 with median age 63 (range 26-89), and 15 (30%) aged ≥70. 17 pts (34%) were female. 28 (56%) pts had DLBCL: 21 (42%) non-GCB, 5 (10%) GCB, 1 (2%) PMBL, and 1 (2%) unknown. 16 (32%) pts had HGBL: 10 (20%) with MYC/ BCL2-R and 6 (12%) with MYC/ BCL6-R. Two (4%) pts each had MCL and EBV-LPD, and 1 pt each had plasmablastic (2%) and Burkitt (2%). Five (10%) pts were treatment naïve and 45 (90%) were relapsed or refractory. 25 (50%) had synchronous CNS and peripheral disease, and 25 (50%) were isolated to the CNS. All relapsed or refractory pts had prior anthracycline, 29 (58%) prior HD-MTX, and 8 (18%) prior CAR-T. Forty-three pts completed the ibrutinib window and were evaluable for responsiveness; 25 (58%) were ibrutinib-responsive and 18 (42%) were ibrutinib-resistant (Figure 1). The overall response rate (ORR) to TEDDI-R (n=28) was 96%, including a CR rate of 71%. The overall response rate to TEDD-R (n=20) was 35%, with a CR rate of 25%. G3 or G4 neutropenia occurred in 43% and 31% of cycles, respectively, and febrile neutropenia occurred in 11%. G3 or G4 thrombocytopenia occurred in 31% and 12% of cycles, respectively, and G3 anemia occurred in 37%. G3 non-hematologic AEs (% pts) included UTI (20%), hypokalemia (22%), sepsis (14%), diarrhea (16%), hypotension (12%), fatigue (10%), anorexia (10%), adrenal insufficiency (10%), and atrial fibrillation (8%). Few G4 AEs were noted. No opportunistic infections (including Aspergillus) were observed. 14 (28%) pts had hand-foot syndrome. After a median follow-up of 24m, the 1-year PFS and OS of all patients were 37.8% and 59.9%, respectively. Pt deaths were due to disease progression (20 pts, 83%), pneumonia (3 pts, 13%), and a cardiac event (1 pt, 4%). The 1-year PFS and OS for pts with ibrutinib-responsive compared to ibrutinib-resistant tumors were 57.0% vs. 16.7% (p=0.0006) and 73.8% vs. 47.9% (p=0.06) (Figure 2). Most (86%) ibrutinib-responsive DLBCL/HGBL tumors were CD10-negative. CD10-negative DLBCL/HGBL pts had an ORR of 77% and a CR rate of 70%. The 1-year PFS and OS for pts with CD10-negative DLBCL/HGBL were 50.5% and 65.0%, respectively. There was no difference in PFS or OS in pts aged <70 versus ≥70 (p=0.87 and p=0.74, respectively). We performed comprehensive genomic profiling on 27 DLBCL/HGBL tumors according to the LymphGen algorithm. 80% (n=4) MCD, 75% (n=3) BN2, 50% (n=2) EZB, and 50% (n=7) unclassified tumors were responsive to ibrutinib. Conclusions: Pts with ibrutinib-responsive SCNSL achieve a high rate of CR to TEDDI-R, and preliminary data suggest these remissions are durable. Most ibrutinib-responsive tumors are CD10-negative and enriched for MCD and BN2. TEDDI-R can be delivered as an outpatient to pts of all ages.

SHP1 loss augments DLBCL cellular response to ibrutinib: a candidate predictive biomarker.

SHP1, a tyrosine phosphatase, negatively regulates B-cell receptor (BCR) signaling. Ibrutinib selectively inhibits BTK and has been approved for the treatment of several types of B-cell lymphomas, but not yet in diffuse large B-cell lymphoma (DLBCL). A phase 3 clinical trial of ibrutinib-containing regimen has been completed to evaluate its activity in subtypes or subsets of DLBCL patients. Although the subtype of activated B-cell like (ABC) DLBCL is characterized by chronic active BCR signaling, only a fraction of ABC-DLBCL patients seem to benefit from ibrutinib-containing regimen. New alternative predictive biomarkers are needed to identify patients who better respond. We investigated if SHP1 plays a role in defining the level of the BCR activity and impacts the response to ibrutinib. A meta-analysis revealed that lack of SHP1 protein expression as well as SHP1 promoter hypermethylation is strongly associated with NHL including DLBCL. On a tissue microarray of 95 DLBCL samples, no substantial difference in SHP1 expression was found between the GCB and non-GCB subtypes of DLBCL. However, we identified a strong reverse correlation between SHP1 expression and promoter methylation suggesting that promoter hypermethylation is responsible for SHP1 loss. SHP1 knockout in BCR-dependent GCB and ABC cell lines increased BCR signaling activities and sensitize lymphoma cells to the action of ibrutinib. Rescue of SHP1 in the knockout clones, on the other hand, restored BCR signaling and ibrutinib resistance. Further, pharmacological inhibition of SHP1 in both cell lines and patient-derived primary cells demonstrate that SHP1 inhibition synergized with ibrutinib in suppressing tumor cell growth. Thus, SHP1 loss may serve as an alternative biomarker to cell-of-origin to identify patients who potentially benefit from ibrutinib treatment. Our results further suggest that reducing SHP1 pharmacologically may represent a new strategy to augment tumor response to BCR-directed therapies. Schematic diagram summarizing the major findings. Left panel. When SHP1 is present and functional, it negatively regulates the activity of the BCR pathway. Right pane. When SHP1 is diminished or lost, cells depend more on the increased BCR signaling and making them vulnerable to BTK inhibitor, ibrutinib. Diagram was generated using BioRender.

Preliminary Result of Phase 1 Trial of Orelabrutinib in Combination with Rituximab, Methotrexate, and Dexamethasone in Patients with Newly Diagnosed Primary CNS Lymphoma Implementing Bayesian Design for Dose-Seeking

Background: Primary central nervous system lymphoma (PCNSL) is an aggressive extranodal subtype of diffuse large B-cell lymphoma (DLBCL). High-dose methotrexate (HD-MTX)-based regimen is the first-line treatment of PCNSL, with or without the anti-CD20 antibody rituximab, remaining to be suboptimal due to unstable response and short effective remission time. Genetic alterations often occur in the chronic active B-cell receptor signaling featured by MYD88 and CD79B which mediate the response to BTK inhibition (BTKi) in PCNSL. Orelabrutinib is a novel, potent, highly selective BTK inhibitor with a high CSF concentration. This study aims to investigate optimal combination dose of Orelabrutinib in combination with Rituximab, Methotrexate, and Dexamethasone (ORMD) as well as safety and efficacy of the regimen. Methods: This was a phase 1, investigator-initiated, dose-escalation study of ORMD, being conducted at Huashan Hospital, Fudan University (ClinicalTrials.gov identifier: NCT05036577). It was planned to enroll 12-15 patients with newly diagnosed PCNSL to the regimen of orelabrutinib and MTX combined with the fixed dose of rituximab(R) and dexamethasone(D). Eligible patients must have histopathologically confirmed PCNSL-DLBCL by biopsy of brain lesions and be aged 18-75 years with adequate organ functions. Patients were treated for 6-8 cycles of induction therapy with 21 days per cycle, receiving rituximab (375mg/m2 on day 1), dexamethasone (10-15mg on d1-d4), MTX (d2, 3.5g/m2 or 5g/m2), and orelabrutinib (once daily, after MTX clearance, 150mg/d, or 200mg/d), followed by orelabrutinib maintenance up to one year among CR/CRu patients. The primary objective was to determine the maximum tolerated dose (MTD) of the combination of orelabrutinib and MTX with R and D and investigate the safety and tolerability of this regimen using Bayesian Optimal Interval (BOIN) waterfall design to determine rule of dose escalation and movement among dose combination matrix to identify MTD contour. DLT was defined by the occurrence of severe toxicities during the first cycle: any grade 4 hematologic toxicity, grade 3 febrile neutropenia and grade 3 thrombocytopenia with hemorrhage, or any grade 3 non-hematologic toxicity that failed to respond to supportive therapy and possibly related to orelabrutinib and/or MTX (assessed according to NCI CTCAE V5.0). The secondary objectives included ORR/CR(u) and PFS/OS. Baseline tissue/CSF samples and sequential CSF/plasma samples were collected for targeted NGS and pharmacokinetics evaluation as exploratory objectives. Results: Eight patients were enrolled from October 2021 to June 2022 as the preliminary analysis set. The median age was 62 year (range: 43-73) and six patients were male. All the patients with histologically confirmed DLBCL (6 non-GCB subtypes and 2 GCB subtypes) had completed at least one cycle of ORMD treatment and were evaluable for DLT. The median cycle of ORMD was one (range: 1-7). No DLT occurred among first three patients in cohort 1 (Orelabrutinib: 150mg/MTX: 3.5g/m2) and dose escalation was allowed to cohort 2 (Orelabrutinib: 200mg/MTX: 3.5g/m2). In cohort 2, 5 patients were dosed in total, and Gr 3 lung infection was observed in patient #4 during cycle 1, was deemed as a DLT. Another four patients were enrolled in the same cohort according to BOIN design algorithm and no DLT was identified, allowing to escalate to cohort 3 (Orelabrutinib:200mg/MTX:5g/m2). No Gr 4 AE had been observed. The most common adverse events were lymphopenia (Gr 3 in 4 case) and hyperglycemia (Gr 3 in 1 case). No atrial fibrillation, hypertension, or bleeding was observed by the cut-off date. The study is ongoing. Further results will be updated as data becomes mature. Conclusions: ORMD regimen preliminarily demonstrated predictable and manageable safety profile in newly diagnosed PCNSL patients with 200mg/d orelabrutinib and 3.5g/m2 MTX to date. MTD contour not reached and further evaluation continues. Longer follow-up is needed regarding efficacy, biomarker, and more safety data Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Establishment and characterization of a new activated B-cell-like DLBCL cell line, TMD12

We report the establishment of a novel activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) cell line, designated as TMD12, from a patient with highly refractory DLBCL. ABC-DLBCL is a subtype with a relatively unfavorable prognosis that was originally categorized using gene expression profiling according to its cell of origin. TMD12 cells were isolated from the pleural effusion of the patient at relapse and passaged continuously in vitro for >4 years. The cells displayed cluster of differentiation (CD)19, CD20, CD22, CD38, human leukocyte antigen-DR isotype, and κ positivity and CD5, CD10, CD23, and λ negativity, as detected using flow cytometric analysis. The chromosomal karyotypic analysis, including the spectral karyotyping method, confirmed t(1;19)(q21:q13.1), del(6q23), gain of chromosome 18, and other abnormalities. Mutation analyses, including whole-exome sequencing, revealed that TMD12 cells harbored mutations in MYD88 and CD79B, indicating an ABC subtype. TMD12 cells exhibited chronic active B-cell receptor signaling and constitutive activation of the nuclear factor κB pathway, which is typically associated with sensitivity to a specific Bruton tyrosine kinase inhibitor, ibrutinib. Intriguingly, TMD12 cells displayed moderate resistance to ibrutinib and lacked activation of Janus kinase/signal transducers and activators of transcription 3 signaling, another hallmark of this DLBCL subtype. Treatment with an inhibitor against tumor progression locus 2 (TPL2), a multifunctional intracellular kinase that is activated particularly downstream of Toll-like receptors or MYD88 and IκB kinase α/β (IKKα/β), suppressed the proliferation of TMD12 cells, implying the possible involvement of the TPL2-p105 pathway in the tumorigenesis of ABC-DLBCL. Because only a limited number of ABC-DLBCL cell lines are currently available, TMD12 cells might provide a useful tool in the search for novel druggable targets for this intractable lymphoma.

Initial Safety Results from a Phase II Study of Acalabrutinib Plus RICE Followed By Autologous Hematopoietic Cell Transplantation and/or Acalabrutinib Maintenance Therapy for Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Background: Patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) often have a poor prognosis despite therapies using second-line chemoimmunotherapy (CIT). Achievement of complete response (CR) with second-line therapy is associated with improved long-term outcomes. Unfortunately, only 25-35% of patients achieve CR with standard CIT regimens alone. The addition of novel targeted agents such as Bruton Tyrosine Kinase inhibitors (BTKi) to second-line therapy may offer improved treatment responses given the importance of B-cell receptor (BCR) signaling in DLBCL. BTK has been shown to be essential for BCR-mediated activation of the NF- κB/Rel family of transcription factors and BCR signaling has been recognized as a key pathway in the pathogenesis of DLBCL. Moreover, NF-κB activity relies upon chronic active BCR signaling in activated B-cell-like DLBCL, which can be potentially blocked by kinase inhibitors targeting BTK. In this study we examine the feasibility and efficacy of adding the BTKi, acalabrutinib (A), to standard second-line therapy to improve disease response in patients with R/R DLBCL. Here we present initial safety and tolerability data for the ongoing study.Study Design and Methods: This is an open-label, prospective phase II trial (NCT03736616). Cohort A is open to R/R DLBCL patients eligible for autologous hematopoietic transplantation (HCT). Cohort B is open to R/R DLBCL patients considered ineligible for autologous HCT. The primary endpoint for cohort A is to estimate the confirmed CR rate (RECIL 2017 criteria) prior to autologous HCT in patients undergoing second-line CIT. The primary endpoint for cohort B is defined as the estimate of one-year progression-free survival in patients undergoing second-line induction and maintenance acalabrutinib therapy.Cohort A receive 2 cycles of standard RICE salvage CIT in combination with acalabrutinib, 100mg BID days 1-21 of a 21-day cycle (RICE+A). After 2 cycles of therapy, patients undergo autologous stem cell mobilization and collection. Patients then receive a 3 rd cycle of RICE in combination with acalabrutinib. PET-CT (PET3) is to be performed on day 15 of cycle 3 to assess response. Patients with CR or partial response (PR) after PET3 proceed to autologous HCT with BEAM conditioning within 28-42 days of PET3. Post-HCT CR patients receive acalabrutinib 100mg BID as maintenance therapy for 12 additional months. Protocol amendment in May 2021 allows for PET assessment (C2D15) prior to autologous stem cell collection (after cycle 3).Cohort B receive 3 cycles of RICE+A in 21-day cycles followed by PET-CT (PET3) on day 15 of cycle 3. Patients without progressive disease at PET3 continue with acalabrutinib maintenance up to 12 additional cycles until disease progression or unacceptable toxicity. Patients demonstrating progressive disease are withdrawn from study treatment but followed for outcomes.Results: Here we report initial safety and tolerability data for the ongoing study with data cutoff July 28, 2021. Twenty-two patients have been screened and 20 patients have received at least 1 cycle of RICE+A. Patient characteristics are shown in Table 1. Fifteen patients (79%) have completed 3 cycles of RICE+A. One patient (5%) discontinued due to an adverse event (AE; recurrent appendicitis), 3 patients (16%) discontinued due to progressive disease, and 1 patient is receiving ongoing RICE+A as of data cutoff. Hematologic AE have been observed in 17 patients (89%) with 74% being Grade 3/4. Amongst these, neutropenia was the most common AE occurring in 47% with all being Grade 3/4, and thrombocytopenia occurring in 32% with all being Grade 3/4. All hematologic AE recovered to baseline or grade 1 in median 7 days. Amongst non-hematologic AE, diarrhea occurred in 21% and 0% were Grade 3/4, nausea 16% with 0% Grade 3/4, and headache in 16% with 0% Grade 3/4. Discontinuation of therapy due to AE occurred in 1 patient (recurrent appendicitis) and dose reduction occurred in 1 patient (Gr 4 neutropenia). Temporary (per protocol) dose holds of A occurred in 9 patients (45%), primarily for cytopenias during concurrent RICE+A. Median duration for dose holds of A was 5.5 days.Conclusion: RICE+A is feasible with manageable primarily hematologic AEs similar to those reported for RICE alone. Enrollment and follow up is ongoing for efficacy endpoints and further toxicity assessment. [Display omitted] DisclosuresBensinger: BMS, Janssen, Poseida, Regeneron, Trillium: Research Funding; Amgen, BMS, Janssen, Sanofi: Speakers Bureau. Glennie: Pharmacyclics/Janssen: Speakers Bureau. Pagel: Pharmacyclics/AbbVie: Consultancy; Incyte/MorphoSys: Consultancy; MEI Pharma: Consultancy; Gilead: Consultancy; Actinium Pharmaceuticals: Consultancy; AstraZeneca: Consultancy; BeiGene: Consultancy; Kite, a Gilead Company: Consultancy; Epizyme: Consultancy. Patel: Bristol Myers Squibb: Consultancy, Speakers Bureau; Janssen: Consultancy; Genentech: Consultancy; BeiGene: Consultancy; TG Therapeutics: Consultancy, Speakers Bureau; Abbvie: Consultancy; Pharmacyclics: Consultancy; Morphosys: Consultancy; Kite Pharma: Consultancy, Speakers Bureau; AstraZeneca: Consultancy, Research Funding, Speakers Bureau; MEI Pharma: Consultancy; ADC Therapeutics: Consultancy; Lilly: Consultancy. OffLabel Disclosure:Acalabrutinib is not FDA approved for treatment of DLBCL and is discussed in the context of an ongoing clinical trial only.

Preliminary Results of a Phase II Study of Methotrexate in Combination with Ibrutinib and Temozolomide (MIT) in Newly Diagnosed Primary CNS Lymphoma

Background: Primary central nervous system lymphoma (PCNSL) is a rare and aggressive subtype of B-cell lymphoma with very poor survival. Genetic alterations often occur in the chronic active B-cell receptor signaling which mediate the response to BTK inhibition (BTKi) in PCNSL. However, the efficacy of high-dose methotrexate as a first-line treatment was suboptimal with a short effective remission time and low response rate. We aimed to investigate the efficacy of induction treatment, high-dose methotrexate in combination with ibrutinib and temozolomide (MIT), in newly diagnosed PCNSL.Methods: Thirty-three patients were planned to be enrolled in this open-label, non-randomized, multi-center phase II prospective clinical study (NCT04514393). Between October 2020 and April 2021, nine newly diagnosed PCNSL patients were enrolled. Eligible patients are ≥18 and ≤75 years of age with adequate organ function and require at least one measurable lesion. The MIT induction treatment included six cycles of high-dose methotrexate (HD-MTX, 3.5 g/m 2, every three weeks), ibrutinib (560 mg/d, after HD-MTX clearance), and temozolomide (150 mg/m 2 d1-d5, every three weeks). After the completion of the six cycles of MIT induction treatment, ASCT consolidation (only for patients < 65 yr) and daily ibrutinib (560 mg/d) were administered up to two years, or until disease progression, intolerable toxicity, or death. Treatment response was evaluated by brain MRI and FDG-PET and/or cerebrospinal fluid (CSF) examination every two cycles of the induction MIT treatment. The primary objective of this study was to evaluate the overall response rate (ORR) of MIT induction treatment for PCNSL defined as the proportion of subjects with complete response (CR) or partial response (PR). The safety and toxicity of MIT treatment were also investigated. No dose reduction or delayed treatment occurred. Baseline tissue samples and sequential CSF/plasma samples were collected and underwent targeted next-generation sequencing (NGS).Results: The median age of the nine enrolled patients was 50 yr (range: 29-68) and six of them were male. Six patients have completed the six cycles of MIT treatment and the rest three had just completed two cycles. Hematologic toxicity was mild, Grade 3-4 neutropenia occurred in one patient. Other side effects such as opportunistic infections (including Aspergillus) were not observed. Eight out of the nine (88.9%) patients achieved CR. While patient #2 progressed and dropped out after two cycles of MIT treatment (Figure A). The mutational profiles of baseline tissue and/or CSF samples revealed that PIM1 and MYD88 mutations were present in 88.9% of the entire cohort followed by HISTHIE and CD78B (6/9, 66.7%). CDKN2A copy number loss was detected in four patients. Four patients with sequential CSF or plasma sampling during MIT treatment were analyzed for dynamic disease monitoring. As shown in the figure A, the clearance of ctDNA in the CSF/plasma samples was observed as early as post one cycle in three CR patients (#1,3,4) and maintained until the latest follow-up. However, the ctDNA remained detectable in the CSF sample of the PD patient (#2), which baseline genetic status shows MYD88 (M232T) mutation alone without CD79B mutations, and with CCND3, CDK4 and ERBB3 gene amplification(Figure B). After a median follow-up of 7 months, the 1-year PFS is 88.9% and the OS is 100%. Other clinical results of this ongoing study (NCT04514393) will be updated.Conclusions: The induction treatment of MIT achieved excellent responses in newly diagnosed PCNSL patients with mild hematologic toxicity. The clearance of ctDNA in CSF/plasma samples was observed during dynamic disease monitoring which was in accord with imageology-based response evaluation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A New Role for the SRC Family Member HCK As a Driver of BCR/SYK Signaling in MYD88 Mutated Lymphomas

A New Role for the SRC Family Member HCK As a Driver of BCR/SYK Signaling in MYD88 Mutated Lymphomas

Acalabrutinib Plus RICE Followed By Autologous Hematopoietic Cell Transplantation and/or Acalabrutinib Maintenance Therapy for Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Background: Patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) often have a poor prognosis despite therapies using second-line chemoimmunotherapy. Achievement of CR with second-line therapy is associated with improved long-term outcomes. Unfortunately, only 25-35% of patients achieve complete response (CR) with RICE chemotherapy alone. The addition of novel targeted agents such as Bruton Tyrosine Kinase inhibitors (BTKi) to second-line therapy may offer improved treatment responses given the importance of B-cell receptor (BCR) signaling in DLBCL. BTK has been shown to be essential for BCR-mediated activation of the NF- κB/Rel family of transcription factors and BCR signaling has been recognized as a key pathway in the pathogenesis of DLBCL. Moreover, NF-κB activity relies upon chronic active BCR signaling in activated B-cell-like DLBCL, which can be potentially blocked by kinase inhibitors targeting BTK. The goal of this study is to examine the feasibility and efficacy of adding the BTKi, acalabrutinib, to standard second-line therapy as a means to improve disease response. Establishing the feasibility of combining acalabrutinib with RICE chemotherapy in autologous hematopoietic cell transplantation (HCT) eligible and HCT ineligible patients with R/R DLBCL may provide the foundation for a larger study of efficacy and long-term outcomes of the combination therapy for patients with R/R DLBCL. Study Design and Methods: The primary objective of this phase 2 trial is to evaluate the tolerability, feasibility, and efficacy of combining acalabrutinib with RICE as second line therapy in R/R DLBCL patients. There are two study cohorts. Cohort A is open to R/R DLBCL patients who are eligible for autologous HCT. Cohort B is open to R/R DLBCL patients who are considered medically ineligible for autologous HCT. The primary endpoint for cohort A is to estimate the confirmed CR rate (RECIL 2017 criteria) prior to HCT in patients undergoing second-line therapy. The primary endpoint for cohort B is defined as the estimate of one-year progression-free survival in patients undergoing second-line induction and maintenance acalabrutinib therapy. Secondary endpoints include assessment of the proportion of patients completing 3 cycles of acalabrutinib with RICE and proceeding with HCT or 2 additional cycles of maintenance acalabrutinib for HCT ineligible patients, overall response rate, incidence of Grade 3/4 adverse events, and incidence of SAEs. Patients in cohort A receive 2 cycles of standard RICE salvage chemoimmunotherapy in combination with acalabrutinib, 100mg BID day 1-21 of a 21 day cycle. After 2 cycles of therapy, patients in cohort A undergo autologous stem cell mobilization and collection. Patients then receive a 3rd cycle of RICE in combination with acalabrutinib. PET-CT (PET3) is performed 14-21 days after day 1 of cycle 3 to assess response. Those patients with CR or partial response (PR) after PET3 proceed to autologous HCT with BEAM conditioning within 28-42 days of PET3. After adequate hematopoietic recovery, patients restart acalabrutinib 100mg BID as maintenance therapy for a period of 12 additional months. Patients in cohort B receive 3 cycles of RICE salvage chemoimmunotherapy in combination with acalabrutinib 100mg BID day 1-21 of a 21-day cycle followed by PET-CT (PET3) 14-21 days after start of Cycle 3. Patients without progressive disease at PET3 continue with acalabrutinib maintenance up to 12 additional cycles until disease progression or unacceptable toxicity. Patients demonstrating progressive disease are withdrawn from study treatment but their outcomes continue to be recorded and will be included in the final data analysis. Historical outcomes from completed, published prospective clinical trials using RICE chemoimmunotherapy serve as a reference for statistical calculations. This trial is currently ongoing and additional information can be found on clinicaltrials.gov NCT listing NCT03736616 Disclosures Bensinger: BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Regeneron: Consultancy, Honoraria, Research Funding, Speakers Bureau. Mawad:Abbvie: Speakers Bureau; Adaptive Biotechnologies: Speakers Bureau. Glennie:Pharmacyclics: Speakers Bureau; Janssen: Speakers Bureau. Patel:Pharmacyclics: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Kite: Consultancy; AstraZeneca: Consultancy, Research Funding, Speakers Bureau; Adaptive Biotechnologies: Consultancy; Genentech: Consultancy, Speakers Bureau; Celgene/BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; BeiGene: Consultancy. OffLabel Disclosure: Acalabrutinib is used an investigational agent for DLBCL in this study.

Preliminary Results of a Response-Adapted Study of Ibrutinib and Isavuconazole with Temozolomide, Etoposide, Liposomal Doxorubicin, Dexamethasone, Rituximab (TEDDI-R) for Secondary CNS Lymphoma

Background: Secondary CNS B-cell lymphomas (SCNSL) are aggressive lymphomas with a very poor prognosis. Genetic subtypes of DLBCL with CNS tropism are enriched for chronic active B-cell receptor signaling and may respond to BTK inhibition (BTKi). CLL, MCL, and transformed lymphomas can also involve the CNS and are BTKi responsive. TEDDI-R achieves durable remissions in relapsed/refractory primary DLBCL of the CNS (PCNSL) but the profile of SCNSL tumors that are ibrutinib-responsive is unknown. We present preliminary results from a response-adapted trial of ibrutinib with TEDD-R in SCNSL. Methods: Pts with aggressive B-cell lymphomas with secondary CNS involvement are eligible if age ≥18 and adequate organ function. Pts must have relapsed after frontline therapy or can be untreated if brain parenchyma involved. Prior BTKi is allowed, but HIVinfection and EBV+ lymphomas are excluded. Baseline tests include brain MRI, FDG-PET brain and body, CSF with flow cytometry, Ommaya, and eye exam. Pts receive isavuconazole starting at 200mg BID x 3d prior to ibrutinib to prevent fungal infections associated with TEDDI-R and then 200mg daily unless ibrutinib stopped. Pts first receive ibrutinib 560mg daily x 14d in a window. If ≥20% reduction after ibrutinib, pts receive TEDDI-R for 4 cycles every 21d with IT cytarabine. Pts with &amp;lt;20% reduction after ibrutinib receive TEDD-R without ibrutinib for 4 cycles every 21d with IT cytarabine. No consolidation or maintenance is permitted. Surveillance for fungal infections includes chest CT after each cycle along with Beta-D glucan and aspergillus galactomannan in blood and CSF. Brain MRI and CT scans are performed after cycles 1, 2, and 4 to determine response. All remissions by MRI are confirmed with FDG-PET and CSF analysis. Surveillance brain MRI and CT scans are q3m for 1y, q4m x 1y, q6m x 1y, then annually. Results: 16 pts with a median age 67 (range 40-79) enrolled between June 2019 and July 2020. 15 (94%) pts had DLBCL comprising 9 (60%) non-GCB, 5 (33%) GCB, and 1 (7%) transformed from MZL. One pt had plasmablastic lymphoma. Eight (50%) pts had a MYC-rearrangement including 4 (25%) with both MYC and BCL2 or BCL6 rearrangements. Eight (50%) pts had isolated CNS disease and 8 (50%) had synchronous CNS and peripheral disease. All pts relapsed after a median of 2 (range 1-4) prior therapies and all (100%) pts received prior anthracycline. Seven pts (44%) had prior CNS prophylaxis and 8 pts (50%) had prior HD-MTX based salvage therapy. Toxicity was evaluated across 35 cycles. G3 and G4 neutropenia occurred in 49% and 29% of cycles, respectively, while febrile neutropenia occurred in 9% of cycles. The median (range) duration of neutropenia was 6 (1-13) days. Five (14%) cycles were complicated by ≥G3 infection, but no opportunistic infections (including Aspergillus) were observed. One pt developed a bacterial infection during cycle 1 and died. G3 and G4 thrombocytopenia occurred in 34% and 23% of cycles, respectively, and 1 pt developed G3 hematuria. G3 mucositis occurred in 9% of cycles and palmar-plantar-erythrodysesthesia led to dose reductions of liposomal doxorubicin in 5 (36%) pts. Of 15 pts who completed the 14d ibrutinib window and were evaluable, 8 (53%) were ibrutinib-responsive and 7 (47%) were ibrutinib-resistant (Figure 1). Clinical responses were concordant across anatomic compartments; of 8 pts with both CNS and peripheral disease, 5 (63%) responded to ibrutinib in both compartments while 3 (37%) did not respond in either compartment. All 8 ibrutinib responders had a non-GCB phenotype and six (75%) achieved CR. One died of treatment-related toxicity after a PR and 1 is still on therapy. Only two (29%) pts with ibrutinib-resistant tumors achieved PR and none have achieved CR. After a median follow-up of 5.1m, a landmark analysis starting after the ibrutinib window demonstrated the PFS for pts with ibrutinib-responsive compared to ibrutinib-resistant tumors was not reached vs. 0.9m (95% CI: 0.1-2m)(p=0.002)(Figure 2). Conclusions: Patients with SCNSL tumors that are ibrutinib-responsive achieve a high rate of complete response to TEDDI-R in both CNS and peripheral disease. Patients with tumors that are ibrutinib-resistant also respond poorly to TEDD-R. Toxicity is mainly hematologic, and no Aspergillus infections have occurred with the use of isavuconazole prophylaxis. Updated clinical results from this ongoing study (NCT03964090) and will be presented at the meeting. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: ibrutinib for use in secondary cns lymphoma as part of a clinical trial

Quantitative Investigation of Pharmacologically Modulated Signaling and Efficacy in ABC DLBCL Using a Systems Pharmacology Model

Introduction The activated B cell like (ABC) subtype of DLBCL has a 40% cure rate with currently available therapies [1] - worse than the rate for germinal center B-cell like (GCB) DLBCL, which highlights the need for ABC subtype-specific treatment strategies. Bruton's tyrosine kinase (BTK) links the activity of the B cell receptor (BCR) to NF-κB and is essential for the survival of ABC cell lines with chronic active BCR signaling. BTK selective covalent inhibitors such as ibrutinib and acalabrutinib are effective in blocking BTK activation and reducing downstream NF-κB pathway activity. However, in ABC DLBCL patients, the observed response rate to ibrutinib is 37% (14/38) and the median PFS is only about 2 months [1]. Many patients show primary resistance to the drug or develop secondary resistance. To improve patient outcomes, a number of combination treatments along with BTK inhibition may be considered. The goal of this work is to integrate data on ABC DLBCL signaling and tumor effect, as well as its pharmacological modulation into one common quantitative systems pharmacology (QSP) modeling framework, to better understand potential efficacy benefits when adding combination partners to a BTK inhibitor. Methods A fit-for-purpose mechanistic model of ABC DLBCL signaling linked to cell cycle and cell death was developed, based on literature curation and in vitro and in vivo data in TMD8 cell lines. TMD8 cells carry activating mutations in the BCR adaptor protein CD79B and in the TLR adaptor protein MYD88. Chronic BCR signaling in ABC DLBCL activates downstream NF-κB through signaling of the BTK as well as the PI3K/AKT/mTOR pathways (Fig. 1). The L265P mutated isoform of MYD88 spontaneously activates NF-κB in a BCR-independent manner. In the cell cycle / cell death part of the model, transition rates were regulated by relevant model variables of signaling pathways (NF-κB, AKT, mTOR activation). Efficacy (tumor size modulation) was implemented through quantitative PK/PD data obtained from TMD8 xenograft studies in CB.17 SCID mice treated with acalabrutinib, as well as mTOR and AKT inhibitors. Results Individual animal tumor size dynamics were studied in control and treatment groups: BTKi (acalabrutinib), AKTi (AZD5363), mTORi (everolimus and AZD2014), as monotherapies and in combinations with acalabrutinib. Interestingly, there was no statistically significant difference in tumor size dynamics, between the control arm and the AKTi monotherapy group. Acalabrutinib monotherapy showed only a moderate decrease in tumor size, with no observation of full tumor rejection. However, acalabrutinib combined with mTORi or AKTi achieved 100% tumor rejection. The model adequately described tumor size dynamics with acalabrutinib and mTORi/AKTi combinations. In order to improve model fitting, we are now taking enhanced activation of the PI3K/AKT/mTOR pathway into consideration, to further evaluate the underlying mechanism for synergy between acalabrutinib and AKTi. Discussion Enhanced AKT activation induction has been observed in BTKi treatment refractory cell lines [2] and may explain primary resistance to, or limited efficacy of BTK inhibitors in some ABC DLBCL patients (while other mechanisms of primary resistance are known). This mechanism may help lymphoma cells to strive, chronically, under BTK inhibitor treatment, while at the same time increase the probability of gaining mutations such as BTK C481S [3], which may further lead to secondary resistance. Combination of an AKT inhibitor with acalabrutinib may help circumvent both primary and secondary (acquired) resistance in ABC-DLBCL.

Reponse-Adapted Study of Ibrutinib with Temozolomide, Etoposide, Doxil, Dexamethasone, and Rituximab (TEDDI-R) in Aggressive B-Cell Lymphomas with Secondary Involvement of the CNS

Background: Aggressive B-cell lymphomas that secondarily involve the CNS (sCNSL) have a grave prognosis and no standard of care. Treatment includes chemotherapy that penetrates the CNS, such as high-dose methotrexate, but are relatively ineffective for extracranial disease. Diffuse large B-cell lymphomas with secondary CNS involvement are enriched for chronic active B-cell receptor signaling, and are responsive to BTK inhibitors. Ibrutinib is also active in other B-cell malignancies that spread to the CNS including MCL and transformed lymphomas. Ibrutinib achieves CNS penetrance and has a high overall response rate in CNS lymphomas, but duration of response is short and curative potential is limited (Grommes et al. 2017). Further, toxicities associated with ibrutinib can compromise outcomes when added to chemotherapy. Precise understanding of molecular determinants of response could enhance patient selection strategies. We developed a novel regimen that combines ibrutinib with temozolomide, etoposide, doxil, dexamethasone, and rituximab (TEDDi-R) for relapsed and refractory PCNSL. TEDDi-R achieved durable remissions in chemotherapy-refractory PCNSL (Roschewski et al. ASH 2018), but has been associated with opportunistic infections, including Aspergillus. Development of TEDDi-R continues with isavuconazole prophylaxis that inhibits clearance of ibrutinib. We opened a novel response-adapted clinical trial designed to study the safety and efficacy of TEDDi-R in sCNSL and elucidate molecular correlates of response to ibrutinib. Study Design and Methods: Phase II study of 29 evaluable patients with recurrent sCNSL [NCT03964090] Patients with untreated aggressive B-cell lymphomas are eligible if CNS lesions involve brain parenchyma Patients are first treated with a 14-day "window" of ibrutinib 560mg monotherapy with concomitant isavuconazole Response adapted: Patients with at least a 20% reduction in bidimensional masses following the 14-day ibrutinib window will receive ibrutinib with chemotherapy (TEDDi-R) for 4 cycles. Patients with less than a 20% reduction during the ibrutinib window will receive TEDD-R (without ibrutinib) for 4 cycles Primary endpoint: progression free survival (PFS) Secondary endpoints: Safety of TEDDi-R and TEDD-R Clinical activity after 14 days of ibrutinib monotherapy Best overall response rate of 4 cycles of TEDDi-R/TEDD-R Duration of response PKs and safety of ibrutinib and TEDDi-R with concomitant isovuconazole prophylaxis Overall survival Translational endpoints: Comprehensive molecular analysis of tumors using gene expression profiling, whole exome sequencing, and RNAseq analysis correlated with outcomeGenotype cell-free DNA in plasma and CSF correlated with clinical response Eligibility: Included Age ≥ 18Prior BTK inhibitors permitted, but patients known refractory to BTK will proceed directly with TEDD-RPatients with CLL or FL that have progressed in the CNS are considered to have transformed to an aggressive B-cell lymphoma and eligibleECOG performance status ≤2Adequate organ functionWilling to take appropriate contraception Excluded HIVInability to take isavuconazoleStatistical methods: Expected median PFS would be less than 4 months based on historical data. The goal is to improve to a median 10 months PFS. With 29 evaluable patients, there would be 90% power to determine a difference between a 4-month median PFS probability and an improved 10-month median PFS, with a one sided 0.05 alpha level test, using the method of Brookmeyer and Crowley. Confidence intervals and PFS probabilities will be reported, but no formal hypothesis test will be undertaken. Summary: This is a phase 2 study of TEDDi-R in aggressive B-cell lymphomas with secondary involvement of CNS that employs a novel response-adapted design for ibrutinib. The aim is to enhance selection of patients likely to benefit from ibrutinib and to investigate the molecular determinants of response to BTK inhibition. The study is open and the first patient has been enrolled. All patients will be treated at the NCI, but the development of TEDDi-R is supported by a consortium of regional academic centers who provide academic input, patient referrals, and data analysis. Current participating centers include Johns Hopkins University, George Washington University, University of Pennsylvania, University of Virginia, University of Pittsburgh and Penn State University. Figure Disclosures Dunleavy: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees. Swinnen:Pharmacyclics: Consultancy; AbbVie: Consultancy. Portell:Kite: Consultancy, Research Funding; Infinity: Research Funding; Roche/Genentech: Research Funding; Acerta/AstraZeneca: Research Funding; TG Therapeutics: Research Funding; Xencor: Research Funding; AbbVie: Research Funding; Pharmacyclics: Consultancy; Janssen: Consultancy; Genentech: Consultancy, Research Funding; Amgen: Consultancy; Bayer: Consultancy; BeiGene: Consultancy, Research Funding. Svoboda:Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Consultancy; Seattle Genetics: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Merck: Research Funding; Incyte: Research Funding; Celgene: Research Funding; Kite: Consultancy; Kyowa: Consultancy. Staudt:Nanostring: Patents &amp; Royalties. OffLabel Disclosure: Ibrutinib used off-label for CNS lymphomas

MARCKS regulates tonic and chronic active B cell receptor signaling.

Tonic or chronic active B-cell receptor (BCR) signaling is essential for the survival of normal or some malignant B cells, respectively. However, the molecular mechanism regulating the strength of these two types of BCR signaling remains unknown. Here, using high-speed high-resolution single-molecule tracking in live cells, we identified that PKCβ, STIM1, and IP3R1/2/3 molecules affected the lateral Brownian mobile behavior of BCRs on the plasma membrane of quiescent B cells, which was correlated to the strength of BCR signaling. Further mechanistic studies revealed that these three molecules influenced BCR mobility by regulating the membrane tethering of MARCKS to the inner leaflet of the plasma membrane. Indeed, membrane-untethered or deficiency of MARCKS significantly decreased, while membrane-tethered or overexpression of MARCKS drastically increased the lateral mobility of BCRs. Functional experiments indicated that the membrane-tethered MARCKS suppressed the survival and/or proliferation in both B-cell tumor cells and mouse primary splenic B cells in vitro and in vivo. Mechanistically, we found that membrane-tethered MARCKS increased BCR lateral mobility, and thus decreased BCR nanoclustering by disturbing the interaction between cortical F-actin and the inner leaflet of the plasma membrane, resulting in the suppression of the strength of both tonic and chronic active BCR signaling. Conclusively, MARCKS is a newly identified molecule regulating the strength of BCR signaling by modulating cytoskeleton and plasma membrane interactions, both in the physiological and pathological conditions, suggesting thatMARCKS is a putative target for drug design.

High Density Lipoprotein-like Nanoparticles Synergize with Akt and Btk Inhibitors to Induce Cell Death in Diffuse Large B Cell Lymphomas

Introduction: We previously reported that our bio-inspired, synthetic high-density lipoprotein-like nanoparticles (HDL NP) induced apoptosis in B cell lymphoma cells expressing scavenger receptor type B1 (SCARB1), the high-affinity receptor for cholesterol-rich HDLs. HDL NPs consist of a 5nm gold nanoparticle core surface functionalized with the HDL-defining apolipoprotein A1 and a phospholipid bilayer, and bind specifically to SCARB1, inducing the efflux of free cholesterol and inhibiting cholesteryl ester influx. SCARB1 is overexpressed in a subset of follicular and diffuse large B cell lymphomas (DLBCL), and resides in cholesterol-rich plasma membrane microdomains called lipid rafts, similar to the B cell receptor (BCR) and its associated signaling kinases. Upon binding to natural HDL, SCARB1 activates a number of pro-survival signaling kinases, including Akt and PI3K. Both Akt and PI3K are also involved in B cell receptor-mediated signaling in germinal center-derived (GC) DLBCL, through tonic BCR signaling, and activated B cell (ABC) DLBCL, through chronic active BCR signaling. Additionally, PI3K was recently shown to play a role in recruitment and activation of Btk, a crucial survival kinase downstream of the BCR. We hypothesized that small molecule inhibitors against pro-survival kinases, specifically Akt and Btk, will synergize with HDL NPs against B cell lymphomas.Methods: Burkitt's lymphoma (Ramos), GC DLBCL (SUDHL4) and ABC DLBCL (TMD8 and HBL-1) cell lines were treated with the Akt inhibitor GDC-0068 or the Btk inhibitor Ibrutinib, in the absence or presence of HDL NPs, and synergy was calculated using the Calcusyn software. Phos-flow was used to assay for changes in the phosphorylation status of Akt and Btk.Results: The Burkitt’s lymphoma and GC DLBCL cell lines were more sensitive to HDL NP induced cell death compared to the ABC DLBCL cell lines (Ramos HDL NP IC50 = 1.5nM; SUDHL4 HDL NP IC50 = 2.1nM; TMD8 HDL NP IC50 = 31.4nM; HBL-1 HDL NP IC50 = 89nM). HDL NPs synergized with GDC-0068 in the Ramos, SUDHL4 and TMD8 cell lines (all combination indexes < 1). Correspondingly, HDL NPs dose-dependently decreased phosphorylation of Akt in Ramos and TMD8 cells. Ibrutinib synergized with the HDL NPs in all cell lines tested (all combination indexes < 1). In TMD8 cells, HDL NPs decreased p-Btk levels comparable to treatment with 10nM Ibrutinib. Addition of the PI3K inhibitor Pilaralisib (XL147) demonstrated mild synergy in the Ramos cell line, but not the SUDHL4, TMD8 or HBL-1 cell lines (all combination index values >1). Treatment of Ramos and SUDHL4 cells with an inhibitor of PTEN, a phosphatase responsible for acting in opposition to PI3K leading to inactivation of Akt, rescued the cells from HDL NP-induced cell death. TMD8 cells treated with the PTEN inhibitor demonstrated a smaller increase in survival when HDL NPs were applied, suggesting that PI3K may not play a major role in HDL NP-induced cell death in activated B cell DLBCLs. PTEN activity is influenced by the level of cholesterol and cholesteryl esters present in the cell, with increasing levels correlating with decreased PTEN activity. Cholesterol levels were higher in the ABC DLBCL cell lines compared to the other B cell lymphoma cell lines. HDL NPs significantly reduced the cholesterol content of Ramos cells, but not the TMD8 or HBL-1 cells, suggesting that the ability of the HDL NPs to alter cellular cholesterol homeostasis correlates with their ability to induce lymphoma cell death.Conclusion: HDL NPs demonstrated synergy with inhibitors to the pro-survival kinases Akt and Btk, suggesting that HDL NPs act to disrupt second messenger signaling pathways in lymphoma cells by directly altering signaling through SCARB1, modulating cellular cholesterol homeostasis, and/or through disruption of membrane raft organization. HDL NPs represent an innovative, targeted therapeutic, with great potential, to add to existing combination chemotherapy regimens. DisclosuresThaxton:Aurasense: Equity Ownership, Patents & Royalties: The patent for the HDL NPs has been licensed to Aurasense, a biotech company co-founded by C. Shad Thaxton..

MiR-17-92 represses PTPROt and PP2A phosphatases and amplifies tonic BCR signaling in DLBCL cells.

B-cell receptor (BCR) signaling plays a pivotal role in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and targeting the BCR pathway is a highly promising therapeutic strategy in this malignancy. The oncogenic microRNA miR-17-92 modulates multiple cellular processes such as survival, proliferation, apoptosis, angiogenesis, and BCR signaling. In the present study, we identified new targets of miR-17-92, PTPROt (protein phosphatase, receptor type O, truncated) and PP2A (protein phosphatase 2A) phosphatases, which regulate the activity of spleen tyrosine kinase (SYK) and AKT, critical components of BCR signal transduction in DLBCL cells. Introduction of miR-17-92 into DLBCL cells dampened the expression of the PTPROt and PP2A regulatory subunits PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) and PPP2R5E (protein phosphatase 2, regulatory subunit B, epsilon isoform) and increased the magnitude of SYK and AKT phosphorylation upon BCR ligation. Finally, we found that miR-17-92 expression modulates response to inhibitors of BCR signaling because downregulation of miR-17-92 increased SYK inhibitor-mediated toxicity in DLBCL cells. Our study reveals novel posttranscriptional regulatory pathways that contribute to the deregulation of BCR signaling and modulate SYK inhibitor activity in DLBCL.

Clonal B cells in Waldenström's macroglobulinemia exhibit functional features of chronic active B-cell receptor signaling.

Waldenström's macroglobulinemia (WM) is a B-cell non-Hodgkin's lymphoma (B-NHL) characterized by immunoglobulin M (IgM) monoclonal gammopathy and the medullary expansion of clonal lymphoplasmacytic cells. Neoplastic transformation has been partially attributed to hyperactive MYD88 signaling, secondary to the MYD88 L265P mutation, occurring in the majority of WM patients. Nevertheless, the presence of chronic active B-cell receptor (BCR) signaling, a feature of multiple IgM+ B-NHL, remains a subject of speculation in WM. Here, we interrogated the BCR signaling capacity of primary WM cells by utilizing multiparametric phosphoflow cytometry and found heightened basal phosphorylation of BCR-related signaling proteins, and augmented phosphoresponses on surface IgM (sIgM) crosslinking, compared with normal B cells. In support of those findings we observed high sIgM expression and loss of phosphatase activity in WM cells, which could both lead to signaling potentiation in clonal cells. Finally, led by the high-signaling heterogeneity among WM samples, we generated patient-specific phosphosignatures, which subclassified patients into a ‘high' and a ‘healthy-like' signaling group, with the second corresponding to patients with a more indolent clinical phenotype. These findings support the presence of chronic active BCR signaling in WM while providing a link between differential BCR signaling utilization and distinct clinical WM subgroups.

Ibrutinib Significantly Prolonged Survival in a Human Burkitt Lymphoma (BL) Xenograft NSG Mouse Model: Ibrutinib May be a Potential Adjuvant Agent in the Treatment of BL

BACKGROUND: Burkitt Lymphoma (BL) represents approximately 40% of all childhood and adolescent non-Hodgkin lymphoma (Miles/Cairo, BJH 2012). Children with relapsed or progressive BL develop chemoimmunotherapy-resistant disease and can rarely be cured with salvage therapy (Cairo et al, JCO 2012). Bruton's tyrosine kinase (BTK) is a regulator of normal B-cell development and is activated upon B-cell receptor (BCR) stimulation. Chronic active BCR signaling through BTK activation can be inhibited by the selective and covalent BTK inhibitor, ibrutinib (Young/Staudt et al, Nat Rev Drug Dicov 2013). Preclinical studies of ibrutinib in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) suggest that its inhibitory effect on cell proliferation is in the range of 1.0uM to 25.0uM (Herman et al, Blood 2011; Cinar et al, Leuk Res 2013), which is higher than the typical plasma concentration range in patients at the recommended dose of 560mg (<0.6uM) (Advani et al, JCO 2013). Despite these relatively high IC50 values in vitro, ibrutinib has been highly effective in the treatment of patients with refractory CLL and MCL (Byrd et al, NEJM 2013 and Wang et al, NEJM 2013). Ibrutinib was initially approved by the FDA (IMBRUVICA, USPI) for patients with MCL in November 2013 and CLL in February 2014, who have received at least one prior therapy. BL, however, is associated with tonic vs chronic active BCR signaling; while, both CLL and MCL have chronic active BCR signaling. These findings suggest that the antitumor activity of ibrutinib in BL, which is associated with tonic BCR signaling, may be less pronounced than in Activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL), CLL or MCL.OBJECTIVES: We hypothesize that ibrutinib may be a potential adjuvant agent in the treatment of BL. Therefore, we investigated the in vivo anti-tumor activity of ibrutinib in BL xenografted NSG mice.METHODS: Raji Burkitt lymphoma (BL) cells (ATCC) were exposed to ibrutinib (0-10uM, generously provided by Janssen R&D LLC) alone and in combination with dexamethasone (1uM) for 5 days and then MTS cell proliferation assay (Promega) was performed. The IC50 values were determined with CompuSynTM software (Chou and Martin, ComboSyn, 2005) based on data derived from MTS assay. Raji BL cells were stably transfected with a firefly luciferase expression plasmid (ffluc-zeo), kindly provided by Laurence Cooper MD, PhD. NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ, 6-8 weeks old) from Jackson laboratory were irradiated (2.5 Gy) and then mice were subcutaneously injected with one million ffluc-zeo tumor cells. Tumor progression and tumor burden were monitored by Bioluminescent Imaging system using the Xenogen IVIS-200 (Caliper Life Sciences). Mice were orally gavaged with either vehicle or ibrutinib (12.5mg/kg) for 10 days (once a day) at post-tumor cells injection. Analysis of survival rates were determined by the Kaplan-Meier method and differences evaluated by log-rank test using the Prism Version 6.0 software.RESULTS: Ibrutinib compared to control induced a significant dose-dependent decrease in cell proliferation in Raji BL cells (5uM, 0.561 ± 0.170, p<0.03, IC50=5.20uM) following 120 hours (5 days) of ibrutinib treatment. Interestingly, we observed a significant decrease in cell proliferation in Raji BL cells (5uM, 0.213 ± 0.078, p<0.002, IC=0.50 ± 0.374uM) following ibrutinib and 1uM dexamethasone combination treatment following 120 hours (5 days) of treatment. We observed a significant decrease of tumor progression by tumor luminescence signal intensity following ibrutinib treated Raji BL xenografted NSG mice at day 20 (12.5mg/kg, p<0.001 and 25mg/kg, p<0.05) and day 25 (12.5mg/kg, p<0.05 and 25mg/kg, p<0.05) compared to control (Figure 1A).Ibrutinib (12.5 mg/kg) treated mice (n=39) significantly prolonged survival compared to vehicle controls (n=28) (p<0.0001) (Figure 1B).CONCLUSIONS: Ibrutinib (12.5mg/kg) significantly decreased tumor progression and significantly increased survival in Raji BL xenografted NSG mice, suggesting that ibrutinib may be a potential adjuvant agent for therapy in the treatment of BL. Future studies will investigate the efficacy of combination therapy with ibrutinib on survival in BL xenografted NOD/SCID mice. [Display omitted] DisclosuresGalardy:Mission Therapeutics: Research Funding.