Resistant Diffuse Large B-cell Lymphoma Research Articles

PI3Kδ activation, IL6 overexpression, and CD37 loss cause resistance to naratuximab emtansine in lymphomas

AbstractCD37-directed antibody and cellular-based approaches have shown preclinical and promising early clinical activity. Naratuximab emtansine (Debio 1562; IMGN529) is an antibody-drug conjugate (ADC) incorporating an anti-CD37 monoclonal antibody conjugated to the maytansinoid DM1 as payload, with activity as a single agent and in combination with rituximab in patients with lymphoma. We studied naratuximab emtansine and its free payload in 54 lymphoma models, correlated its activity with CD37 expression, characterized 2 resistance mechanisms, and identified combination partners providing synergy. The activity, primarily cytotoxic, was more potent in B- than T-cell lymphoma cell lines. After prolonged exposure to the ADC, 1 diffuse large B-cell lymphoma (DLBCL) cell line developed resistance to the ADC due to the CD37 gene biallelic loss. After CD37 loss, we also observed upregulation of interleukin-6 (IL6) and related transcripts. Recombinant IL6 led to resistance. Anti-IL6 antibody tocilizumab improved the ADC’s cytotoxic activity in CD37+ cells. In a second model, resistance was sustained by a PIK3CD activating mutation, with increased sensitivity to PI3Kδ inhibition and a functional dependence switch from MCL1 to BCL2. Adding idelalisib or venetoclax overcame resistance in the resistant derivative and improved cytotoxic activity in the parental cells. In conclusion, targeting B-cell lymphoma with the naratuximab emtansine showed vigorous antitumor activity as a single agent, which was also observed in models bearing genetic lesions associated with inferior outcomes, such as MYC translocations and TP53 inactivation or R-CHOP (rituximab, cyclophosphamide, doxorubicin, Oncovin [vincristine], and prednisone) resistance. Resistant DLBCL models identified active combinations of naratuximab emtansine with drugs targeting IL6, PI3Kδ, and BCL2.

A Bruton tyrosine kinase inhibitor-resistance gene signature predicts prognosis and identifies TRIP13 as a potential therapeutic target in diffuse large B-cell lymphoma

Bruton tyrosine kinase inhibitor (BTKi) combined with rituximab-based chemotherapy benefits diffuse large B-cell lymphoma (DLBCL) patients. However, drug resistance is the major cause of relapse and death of DLBCL. In this study, we conducted a comprehensive analysis BTKi-resistance related genes (BRRGs) and established a 10-gene (CARD16, TRIP13, PSRC1, CASP1, PLBD1, CARD6, CAPG, CACNA1A, CDH15, and NDUFA4) signature for early identifying high-risk DLBCL patients. The resistance scores based on the BRRGs signature were associated with prognosis. Furthermore, we developed a nomogram incorporating the BRRGs signature, which demonstrated excellent performance in predicting the prognosis of DLBCL patients. Notably, tumor immune microenvironment, biological pathways, and chemotherapy sensitivity were different between high- and low-resistance score groups. Additionally, we identified TRIP13 as a key gene in our model. TRIP13 was found to be overexpressed in DLBCL and BTKi-resistant DLBCL cell lines, knocking down TRIP13 suppresses cell proliferation, promotes cell apoptosis, and enhances the apoptosis effect of BTKi on DLBCL cells by regulating the Wnt/β-catenin pathway. In conclusion, our study presents a novel BRRGs signature that could serve as a promising prognostic marker in DLBCL, and TRIP13 might be a potential therapeutic target for resistant DLBCL.

Rupatadine-inhibited OTUD3 promotes DLBCL progression and immune evasion through deubiquitinating MYL12A and PD-L1

The obstacle to effectively treating Diffuse Large B-cell Lymphoma (DLBCL) lies in the resistance observed toward standard therapies. Identifying therapeutic targets that prove effective for relapsed or refractory patients poses a significant challenge. OTUD3, a deubiquitinase enzyme, is overexpressed in DLBCL tissues. However, its role in DLBCL has not been investigated. Our study has brought to light the multifaceted impact of OTUD3 in DLBCL. Not only does it enhance cell survival through the deubiquitination of MYL12A, but it also induces CD8+ T cell exhaustion within the local environment by deubiquitinating PD-L1. Our findings indicate that the OTUD3 inhibitor, Rupatadine, exerts its influence through competitive binding with OTUD3. This operation diminishes the deubiquitination of both MYL12A and PD-L1 by OTUD3. This research unveils the central and oncogenic role of OTUD3 in DLBCL and highlights the potential clinical application value of the OTUD3 inhibitor, Rupatadine. These findings contribute valuable insights into addressing the challenges of resistant DLBCL cases and offer a promising avenue for further clinical exploration.

Abstract P23: RHOA inactivation subverts IFN-γ response and promotes antigen escape in CAR-T resistant diffuse large B-cell lymphoma

Abstract P23: RHOA inactivation subverts IFN-γ response and promotes antigen escape in CAR-T resistant diffuse large B-cell lymphoma

Abbv-101, a Highly Potent and Selective Clinical Stage Bruton Tyrosine Kinase Degrader for the Treatment of B-Cell Malignancies

Bruton tyrosine kinase (BTK), a clinically validated target for various B-cell malignancies, plays a critical role in regulating cell growth, adhesion and homing to key lymphoid tissues that provide a microenvironment favorable to cancer cells. Despite the success of approved covalent BTK inhibitors in diseases such as CLL, most patients do not achieve complete response with monotherapy and eventually relapse, often via the emergence of the BTK-C481S mutation. Additionally, covalent BTK inhibitors have not shown sufficient differentiation from standard-of-care (SoC) in B-cell malignancies such as diffuse large B cell lymphoma (DLBCL) to warrant approval to date. To address these unmet needs, a next-generation orally bioavailable BTK degrader ABBV-101 has been developed. Unlike BTK inhibitors that solely bind to and impede the catalytic domain of BTK, ABBV-101 eliminates the protein in a highly selective fashion. This degradation mechanism targets both the catalytic and scaffolding functions of BTK, and thus may enable deeper and more durable responses in patients with B-cell malignancies. In preclinical studies, the BTK degrader ABBV-101 has shown potent in vitro and in vivo efficacy in BCR pathway-dependent leukemia/lymphoma models, including covalent BTK inhibitor resistant BTK-C481S systemic mouse CLL and human DLBCL models. ABBV-101 degrades BTK-WT and BTK-C481S with similar sub-nanomolar potency in the human DLBCL cell line TMD8 which translates to potent cellular growth inhibition. In addition to the BTK-C481S mutation, ABBV-101 demonstrates similar potent activity against novel BTK mutations associated with resistance to reversible BTK inhibitors in the clinic, an emerging unmet need. ABBV-101 induces complete tumor regression in the BTK-C481S TMD8 DLBCL xenograft model. A spontaneous mouse CLL model carrying BTK-C481S mutation was developed through crossing BTK-C481S knock-in mice with the Emu-TCL1 transgenic mice in the C57BL/6 background. ABBV-101 completely inhibits BTK-C481S Emu-TCL1 CLL burden increase in the blood compartment and reduces CLL burden in the spleen and lymph nodes, whereas covalent BTK inhibitors show no activity. Further, ABBV-101 induces complete tumor regressions in multiple non-GCB DLBCL PDX models, demonstrating deeper and more durable response than covalent and reversible inhibitors. Combination with a BCL-2 inhibitor enhances the efficacy of ABBV-101 in CLL and DLBCL models. Kinome and global proteomics profiling showed ABBV-101 to be highly selective, a designed feature that may enable favorable tolerability in patients. ABBV-101 is currently in phase 1 clinical trial for a variety of B-cell malignancies. ClinicalTrials.gov identifier: NCT05753501

Inhibition of USP1 reverses the chemotherapy resistance through destabilization of MAX in the relapsed/refractory B-cell lymphoma

The patients with relapsed and refractory diffuse large B-cell lymphoma (DLBCL) have poor prognosis, and a novel and effective therapeutic strategy for these patients is urgently needed. Although ubiquitin-specific protease 1 (USP1) plays a key role in cancer, the carcinogenic effect of USP1 in B-cell lymphoma remains elusive. Here we found that USP1 is highly expressed in DLBCL patients, and high expression of USP1 predicts poor prognosis. Knocking down USP1 or a specific inhibitor of USP1, pimozide, induced cell growth inhibition, cell cycle arrest and autophagy in DLBCL cells. Targeting USP1 by shRNA or pimozide significantly reduced tumor burden of a mouse model established with engraftment of rituximab/chemotherapy resistant DLBCL cells. Pimozide significantly retarded the growth of lymphoma in a DLBCL patient-derived xenograft (PDX) model. USP1 directly interacted with MAX, a MYC binding protein, and maintained the stability of MAX through deubiquitination, which promoted the transcription of MYC target genes. Moreover, pimozide showed a synergetic effect with etoposide, a chemotherapy drug, in cell and mouse models of rituximab/chemotherapy resistant DLBCL. Our study highlights the critical role of USP1 in the rituximab/chemotherapy resistance of DLBCL through deubiquitylating MAX, and provides a novel therapeutic strategy for rituximab/chemotherapy resistant DLBCL.

Re-Wiring BCL-2 Family Anti-Apoptotic Protein Dependencies through Modulating Phosphorylation to Re-Sensitize Venetoclax-Resistant Lymphoid Malignancies to BCL-2 Inhibition

Introduction Resistance to apoptosis is a hallmark of cancer, and modulation of BCL-2 family proteins is an important mediator of such resistance in hematologic malignancies. Despite the clinical efficacy of the BCL-2 inhibitor venetoclax (VEN), prolonged treatment may lead to resistance, such as the BCL2 G101V mutation (Blombery et al, Blood, 2020); however, over half of VEN resistant cases are not explained by known genetic mechanisms. Phosphorylation of BCL-2 at serine-70 (S70pBCL2) or of MCL-1 at threonine-163 (T163pMCL1) have been shown to increase sequestration of the pro-apoptotic protein BAX (Deng et al, JNCI, 2000) and stabilize the level of anti-apoptotic protein MCL-1 (Wang et al, Mol Cancer, 2014), respectively. We hypothesized that the increase in post-translational modifications of BCL-2 family members, in particular S70pBCL2 and T163pMCL1, are novel mechanisms of functional VEN resistance in lymphoid malignancies. We further hypothesized that the FDA-approved phosphatase activator drug FTY720 (fingolimod) would de-phosphorylate these BCL-2 family members and thereby re-sensitize malignant lymphoid cells to VEN-induced apoptosis. Methods A VEN resistant diffuse large B-cell lymphoma cell line (OCI-Ly1-R) as well as peripheral blood mononuclear cells from 12 previously untreated CLL patients co-cultured with human stromal NK-Tert cells were treated ex vivo with VEN +/- FTY720. A VEN sensitive cell line (OCI-Ly1-S) was treated with VEN +/- a phosphatase inhibitor okadaic acid (OA). Western blot was used to evaluate changes in S70pBCL2 and T163pMCL1 protein levels. BH3 profiling via flow cytometry was performed to determine the survival dependence on anti-apoptotic BCL-2 family members via cytochrome c release in response to specific BH3-only peptides and drugs such as VEN applied directly to mitochondria (Ryan et al, Biol Chem, 2016). Cell viability assays (CellTiter-Glo, Trypan Blue and Annexin/Hoechst) were employed to investigate the effects of FTY720 on OCI-Ly1-R resistance to VEN. The BCL-2-BAX interaction was investigated using co-immunoprecipitation in VEN resistant and sensitive cells following treatment with VEN +/- FTY720. T-test, ANOVA and multiple comparison with a statistical significance set at 2-tailed p ≤ 0.05 were used. Results OCI-Ly1-R cells displayed higher S70pBCL2, T163pMCL1 and MCL-1 expression compared to OCI-Ly1-S cells. Notably, the increase in S70pBCL2 was associated with reduced response of VEN-mediated BCL-2-BAX dissociation, while the increase in T163pMCL1 was accompanied by enhanced MCL-1 protein expression. Using BH3 profiling, we found that the increase in S70pBCL2, T163pMCL1 and MCL-1 expression were functionally associated with a decrease in BCL-2 survival dependence (-79.1%, 1μM VEN, P < 0.0001) and an increase in MCL-1 dependence (+52%, 10μM MS1, P < 0.0001) (Fig. A). The addition of FTY720 reversed these observations in OCI-Ly1-R cells, where we observed a decrease in S70pBCL2, T163pMCL1 and MCL-1 protein expression, BCL-2 and BAX interaction, as well as a "re-wired" functional dependence toward BCL-2 (-21.6% 10μM MS1, +27.9% 1μM VEN, P < 0.0001) (Fig. B). Importantly, pre-treatment with FTY720 re-sensitized OCI-Ly1-R cells to VEN-induced cell death (+56.1%, P = 0.0001) (Fig. C). Conversely, treatment with a phosphatase inhibitor, OA, led to an increase in S70pBCL2, T163pMCL1 and MCL-1 expression as well as reduced late death of OCI-Ly1-S cells (-60%, P = 0.0018). We validated our cell line results in primary CLL cells, and again the combination of FTY720 and VEN similarly reduced S70pBCL2, T163pMCL1 and MCL-1 expression, increased BCL-2 dependence, and enhanced VEN-induced cell death (+23.6%, P < 0.0001). Conclusion Increased S70pBCL2 and T163pMCL1 are associated with VEN resistance, in part by inhibiting VEN-induced BCL-2-BAX dissociation and switching the functional survival dependence from BCL-2 to MCL-1. FTY720 re-sensitizes VEN resistant cells by reducing S70pBCL2, T163pMCL1 and MCL-1 expression, dissociating BAX from BCL-2 and "re-wiring" the survival dependence to BCL-2. These preclinical findings support the exploration of this strategy clinically in patients with VEN resistant lymphoid malignancies. Disclosures Davids: Sunesis: Consultancy; AbbVie: Consultancy; Surface Oncology: Research Funding; Genentech: Consultancy, Research Funding; Eli Lilly: Consultancy; Celgene: Consultancy; AstraZeneca: Consultancy, Research Funding; BeiGene: Consultancy; Ascentage Pharma: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Zentalis: Consultancy; Novartis: Consultancy, Research Funding; Gilead Sciences: Consultancy; Bristol Myers Squibb: Research Funding; Janssen: Consultancy; MEI Pharma: Consultancy, Research Funding; Syros Pharmaceuticals: Consultancy; Merck: Consultancy; Research to Practice: Honoraria.

Activation of unfolded protein response overcomes Ibrutinib resistance in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most widespread type of non-Hodgkin lymphoma (NHL). As the most aggressive form of the DLBCL, the activated B-cell-like (ABC) subtype is often resistant to standard chemotherapies. Bruton's tyrosine kinase (BTK) inhibitor ibrutinib provides a potential therapeutic approach for the DLBCL but fails to improve the outcome in the phase III trial. In the current study, we investigated the molecular mechanisms underlying ibrutinib resistance and explored new combination therapy with ibrutinib. We generated an ibrutinib-resistant ABC-DLBCL cell line (OCI-ly10-IR) through continuous exposure to ibrutinib. Transcriptome analysis of the parental and ibrutinib-resistant cell lines revealed that the ibrutinib-resistant cells had significantly lower expression of the unfolded protein response (UPR) marker genes. Overexpression of one UPR branch-XBP1s greatly potentiated ibrutinib-induced apoptosis in both sensitive and resistant cells. The UPR inhibitor tauroursodeoxycholic acid (TUDCA) partially reduced the apoptotic rate induced by the ibrutinib in sensitive cells. The UPR activator 2-deoxy-D-glucose (2-DG) in combination with the ibrutinib triggered even greater cell growth inhibition, apoptosis, and stronger calcium (Ca2+) flux inhibition than either of the agents alone. A combination treatment of ibrutinib (15 mg·kg-1·d-1, po.) and 2-DG (500 mg/kg, po, b.i.d.) synergistically retarded tumor growth in NOD/SCID mice bearing OCI-ly10-IR xenograft. In addition, ibrutinib induced the UPR in the sensitive cell lines but not in the resistant cell lines of the DLBCL. There was also a combined synergistic effect in the primary resistant DLBCL cell lines. Overall, our results suggest that targeting theUPR could be a potential combination strategy to overcome ibrutinib resistance in the DLBCL.

Abstract 2404: MDM2 inhibitor Idasanutlin potentiate therapeutic agents in Diffuse Large B-Cell Lymphoma by targeting MDM2 and XIAP

Abstract Purpose: In patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL), resistance to rituximab confers chemotherapy-resistance and poor response to salvage regimens. MDM2 is an E3 ligase which regulates the degradation of multiple cellular protein targets, but is known best as the major negative regulator of the tumor suppressor, p53. Recently, we demonstrated that MX69, a MDM2 inhibitor, re-sensitized resistant DLBCL cell lines to chemotherapy agents or small inhibitors (i.e. Venetoclax) in vitro by blocking the MDM2 protein-XIAP RNA interaction, led to both MDM2 and XIAP degradation, thus establishing the feasibility of this approach for overcoming rituximab and chemotherapy resistance in DLBCL. We now report the activity of Idasanutlin, an investigational Nutlin-family MDM2 antagonist, in pre-clinical models of R-chemo resistant B-cell lymphoma. Methods: We used a panel of rituximab sensitive and R-chemo resistant B-cell lymphoma cell lines representing activated B-cell-like (ABC) DLBCL (TMD8, U2932); germinal center B-cell like (GCB) DLBCL (RL, RL 4RH, OCILy2); double hit DLBCL (DHL4, DHL6, DOHH2, VAL, ROS50), and Burkitt's lymphoma (Raji and Raji 4RH). Cells were exposed to escalating doses of Idasanutlin as single agent and in combination with various chemotherapeutic agents and small molecule inhibitors for 24, 48 and 72 hrs. Differences in cell viability were evaluated by PrestoBlue. IC50 was calculated by GraphPad and Coefficient of synergy was calculated using CalcuSyn. Low mitochondrial potential was detected by staining cell with DiOC6 followed by flow cytometric analysis. Western blot was used to detect changes in MDM2, XIAP and PUMA expression. Results: In vitro exposure to Idasanutlin demonstrated dose- and time-dependent cell death in DLBCL cell lines, IC50 ranged from 0.7uM to 63.07uM at 48h. Idasanutlin at the dose of 1uM was able to disrupt mitochondria and lowered the mitochondrial membrane potential in both ABC and GCB cell lines at 48 h. Idasanutlin reduced the expression of MDM2 in TMD8, U2932, VAL, OCILy2 DHL4 and ROS50 cell lines. XIAP expression was reduced in VAL and DHL4. PUMA, the downstream product of p53 activation, was increased after idasanutlin exposure in all cell lines. Idasanutlin demonstrated significant synergy with chemotherapeutic reagents (etoposide and vincristine), proteasome inhibitors (carfilzomib and ixazomib), Bcl-2 inhibitor venetoclax and Bruton tyrosine kinase inhibitor ibrutinib. Conclusion: Idasanutlin showed promising cell-of-origin agnostic anti-tumor activity as a single agent and in combination with several small molecule inhibitors in pre-clinical models of DLBCL. It decreased MDM2 and XIAP protein level and induced PUMA expression. Its unique mechanism of action through p53 modulation makes it an attractive partner for use in a variety of clinical settings in B-cell lymphomas. Citation Format: Juan J. Gu, Samuel Thompson, Cory Mavis, Pallawi Torka, Francisco Hernandez-Ilizaliturri. MDM2 inhibitor Idasanutlin potentiate therapeutic agents in Diffuse Large B-Cell Lymphoma by targeting MDM2 and XIAP [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2404.

PI3K/AKT inhibition reverses R-CHOP resistance by destabilizing SOX2 in diffuse large B cell lymphoma.

Up to one-third of diffuse large B cell lymphoma (DLBCL) patients eventually develop resistance to R-CHOP regimen, while the remaining therapeutic options are limited. Thus, understanding the underlying mechanisms and developing therapeutic approaches are urgently needed.Methods: We generated two germinal center B cell-like (GCB) and activated B cell-like (ABC) subtype R-CHO resistant DLBCL cell lines, of which the tumor-initiating capacity was evaluated by serial-transplantation and stemness-associated features including CD34 and CD133 expression, side population and ALDH1 activity were detected by flow cytometry or immunoblotting. Expression profiles of these resistant cells were characterized by RNA sequencing. The susceptibility of resistant cells to different treatments was evaluated by in vitro CytoTox-glo assay and in tumor-bearing mice. The expression levels of SOX2, phos-AKT, CDK6 and FGFR1/2 were detected in 12 R-CHOP-resistant DLBCL clinical specimens by IHC.Results: The stem-like CSC proportion significantly increased in both resistant DLBCL subtypes. SOX2 expression level remarkably elevated in both resistant cell lines due to its phosphorylation by activated PI3K/AKT signaling, thus preventing ubiquitin-mediated degradation. Further, multiple factors, including BCR, integrins, chemokines and FGFR1/2 signaling, regulated PI3K/AKT activation. CDK6 in GCB subtype and FGFR1/2 in ABC subtype were SOX2 targets, whose inhibition potently re-sensitized resistant cells to R-CHOP treatment. More importantly, addition of PI3K inhibitor to R-CHOP completely suppressed the tumor growth of R-CHO-resistant DLBCL cells, most likely by converting CSCs to chemo-sensitive differentiated cells.Conclusions: The PI3K/AKT/SOX2 axis plays a critical role in R-CHOP resistance development and the pro-differentiation therapy against CSCs proposed in this study warrants further study in clinical trials for the treatment of resistant DLBCL.

Mutational Dynamics and Evolutionary Divergence in DLBCL: A Call for Relapse Sampling

Introduction Relapses of diffuse large B-cell lymphoma typically occur within 2-3 years and only 10% of these patients reach a 3-year progression-free survival compared to 65% at diagnosis. Our ability to distinguish patients at risk for relapse remains based on clinical staging. We hypothesized that identifying genetic alterations in serial tumour biopsies at diagnosis and relapse would improve our ability to identify high-risk patients, make therapeutic selections and reveal molecular markers for chemo-immunotherapy resistant tumours. However, relatively few relapsed/refractory biopsies have been sequenced. A unique, clinically annotated, Nordic DLBCL cohort was used to identify significantly mutated genes, assess potential driver genes, comprehensively examine clonal evolution, and gauge the importance of clinical relapsed sampling. Methods To address the lack of information on the molecular foundations of relapsed/refractory DLBCL, we performed whole exome sequencing (WES) on 42 DLBCL cases, with 34% representing relapsed/refractory biopsies and 13 serially sample cases. Enriched with relapsed/refractory diffuse large B-cell lymphoma cases, we performed multiple computational analyses to identify significantly mutated genes (MutSig2CV), mutational signatures (NMF and DeConstructsSig), driver genes (IntOgen and CADD), clonal evolution architecture (SciClone and ClonEvol), druggable gene analysis (DGIdb), and HLA-inference and mutation calling (Polysolver). Results Clonal evolution analysis of 13 paired diagnostic and relapsed biopsies revealed that relapsed/refractory biopsies have remarkable similarities to diagnostic biopsies and often present with late divergent clonal evolution of the tumor. Mutational analysis of 18 serially sampled tumors determined that in the majority of cases druggable oncogenic variants do arise at relapse. In addition, time to relapse correlated with divergence of mutations from the diagnostic biopsy. In addition to being identified as a significantly mutated gene, mutations in HLA-A had an increased incidence in cases that ultimately relapsed. This result led to an in-depth investigation into the mutational prevalence, timing, impact on prognosis, and loss of heterozygosity in the human leukocyte antigen (HLA) haplotypes of relapsed/refractory DLBCL. HLA-A mutagenesis and loss of heterozygosity was discovered as mechanisms of immune evasion in cases that go on to relapse from R-CHOP like therapies (Figure 1). Conclusions Our results yield insight into the development of chemo-immunotherapy resistant diffuse large B-cell lymphoma, and highlight the clinical importance of sampling relapsed biopsies. Analysis of immune evasion through MHC Class I/II, specifically HLA-A, may provide better characterization of patients for relapse prediction. In the age of personalized medicine it will be instrumental to determine if relapsed biopsies offer additional insight for salvage therapy treatment. Divergence of biopsies, as characterized by shared genomic mutations, increase with time and the majority of cases present with new alterations in druggable genes post-therapy. Disclosures Leppa: Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy; Bayer: Research Funding. Holte:Novartis: Honoraria, Other: Advisory board.

Targeting MDM2 and XIAP By Idasanutlin in Diffuse Large B-Cell Lymphoma

Purpose: The combination of rituximab and chemotherapy has improved overall survival of diffuse large B-cell lymphoma (DLBCL) patients in recent decades. Relapsed/refractory DLBCL patients previously treated with rituximab-containing regimen had significantly poorer response to salvage therapy. To study the mechanisms of this resistance, our laboratory developed several rituximab resistant cell lines. Previous study from our group found demonstrated an aberrant imbalance in the levels of pro- and anti-apoptotic proteins in these cell lines, including Bak/Bak, Mcl-1/BCLxL/Survivin and inhibitor of apoptosis (IAP) family proteins resulting in acquired chemotherapy resistance. MDM2 is an E3 ligase which regulates the degradation of multiple cellular protein targets such as pRB, HIF-1, p73, NF-kB, and E2F1 as well as FOXO3a. It is also the major negative regulator of p53. Recently, we found that MX69 (a MDM2 inhibitor), which blocks the MDM2 protein-XIAP RNA interaction, led to both MDM2 and XIAP degradation, and induced apoptosis-dependent cell killing activity. Moreover, MX69 re-sensitized resistant DLBCL cell lines to chemotherapy agents or small inhibitors (i.e. Venetoclax) in vitro. However, MX69 cannot be used clinically stressing the need to use a clinically available MDM2 inhibitor. To this end, we evaluated Idasanutlin, an investigational Nutlin family MDM2 antagonist, in B-cell lymphoma pre-clinical models. Methods: We used a panel of different DLBCL subtypes cell lines including activated B-cell lymphoma cell lines (TMD8, U2932); germinal center B-cell lymphoma (DOHH2, VAL, OCILY2, DH4, DH6, ROS50); rituximab-sensitive cell lines (Raji and RL); and rituximab/-resistant cell lines (Raji 4RH and RL 4RH). Cells were exposed to escalating doses of Idasanutlin (1nM-100µM) without or with other chemotherapeutic agents for 24, 48 and 72 hrs. Differences in cell viability were evaluated utilizing PrestoBlue. IC50 was calculated by GraphPad and Coefficient of synergy was calculated using CalcuSyn. Low mitochondrial potential was detected by staining cell with DiOC6 10ng/ml for 30 minutes and followed by flow cytometric analysis. Western blot was used to detect the changes of MDM2, XIAP and PUMA expression before and after exposure to Idasanutlin 1uM for 24h. Results:In vitro exposure Idasanutlin to DLBCL cell lines demonstrated a dose- and time-dependent cell death. IC50 dosage of the cells was ranged from 0.7uM to 63.07uM at 48h. Low dose of Idasanutlin (1uM) was able to disrupt mitochondria and induced low mitochondrial membrane potential in both ABC and GCB cell lines. At molecular level, Idasanutlin reduced expression level of MDM2 in TMD8, U2932, VAL, OCILy2 DHL4 and ROS50 cell lines. XIAP was reduced in Val and DHL4. Meanwhile, PUMA, the downstream of p53 activation, was increased after Idasanutlin exposure. Idasanutlin enhanced the anti-tumor activity of proteasome inhibitors (carfilzomib, ixazomib), Bcl-2 inhibitor venetoclax and Bryton kinase inhibitor ibrutinib. Conclusion: Idasanutlin showed potent anti-tumor activity as a single agent in DLBCL pre-clinical setting. Idasanutlin was able to decrease cellular mitochondrial membrane potential. At molecular level, Idasanutlin decreased MDM2 and XIAP protein level and induced PUMA expression. Moreover, Idasanutlin enhanced anti-tumor activities of other small inhibitors. Taking together, Idasanutlin could be used as a novel and promising drug in the clinical setting of DLBCL with relapsed/refractory disease. Disclosures No relevant conflicts of interest to declare.

PS1321 INHIBITION OF NOTCH SIGNALING RESTORES SENSITIVITY TO IMMUNOCHEMOTHERAPY IN AN IN VITRO MODEL OF ACQUIRED RESISTANCE TO R‐CHOP

Background:Diffuse large B‐cell lymphoma (DLBCL) accounts for almost 40% of all lymphomas. One‐third of DLBCL patients either initially fail to respond to Rituximab plus anthracycline based therapy CHOP (R‐CHOP) or suffer a relapse (i.e. acquired resistance). The Notch pathway is involved in cell proliferation, differentiation and survival in a wide range of cancers.Aims:We investigated the role of Notch signaling in an in vitro model of acquired resistance to R‐CHOP treatment.Methods:We developed an in vitro model of acquired R‐CHOP resistance using different DLBCL cell lines (SU‐DHL‐4, SU‐DHL‐10 for GCB‐like subtype; RIVA and U2938 for ABC‐like subtype). Using gene expression microarrays, we screened parental and R‐CHOP resistant cell lines to identify putative genes responsible for acquired resistance. Notch1/2 and JAG1 expression levels were validated by qPCR and flow cytometry in R‐CHOP resistant U2932 cells. We treated U2932 cells with a neutralising anti‐JAG1 antibody (clone J1–65D, 10ug/ml) or with the γ‐secretase inhibitor dibenzazepine (DBZ, 100 nM). We then evaluated cell viability after 72 h of R‐CHOP treatment at 400 ng/ml (corresponding to the GI50 of the resistant clones).Results:Our microarray analysis showed many genes were differentially expressed. We focused particularly on the U2932 cell line, where overexpression of NOTCH1, NOTCH2 and JAG1 were independently confirmed by qPCR and flow cytometry. We evaluated the viability of U2932 after 72 h treatment with R‐CHOP and we found that the treatment of the resistant clones with DBZ or anti‐JAG1 restored sensitivity to RCHOP, compared to DMSO (vehicle) or IgG controls.Summary/Conclusion:We have demonstrated that blocking of Notch signaling, either by preventing JAG1 ligand binding or impeding the cleavage of the intracellular Notch domain, can restore sensitivity to R‐CHOP at levels comparable to the parental control. These preliminary results may suggest Notch signaling as new therapeutic target for resistant DLBCL.

The Fate of Patients with Refractory Diffuse Large B Cell Lymphoma (DLBCL)

Background:The outcome of DLBCL patients is improving, it however seems to be very poor for those who are refractory to the therapy. We have decided to analyze this group of patients.Methodology:As part of an observational clinical study NiHiL (GovTrial No NCT03199066) we identified the patients treated in the first line by R-CHOP like immunochemotherapy who met at least one criterium: 1. refractory (stable disease - SD or progression) on 1st line therapy (1st l- R), 2. refractory (SD or progression) on salvage (platinum based regimen) (Salv-R), 3. refractory to or relapse/progression within 12 months after ASCT (ASCT-R/R). There were 210 patients diagnosed in the period 2001-2017 who fulfilled the criteria. Progression-free survival (PSF) and overall survival (OS) were estimated from the time of determination of a refractory disease.Results:The cohort consisted of 163 patients primarily resistant to the first-line therapy, 31 patients were resistant to the salvage therapy and 16 patients progressed within 12 months after ASCT . At the time of the diagnosis, the median age was 65 years (22-91) (the same as at the refractory disease), 54% were males, 74% had advanced clinical stage (III+IV), 68% had IPI >3, 77% had above-normal LDH, and 45% had a tumor mass >10cm.The OS from the time of determination of refractory disease was 0.53 years, median PFS was 0.29 years. Patients under 65 years had median survival 0.8 years compared to 0.4 years in the group of patients above 65 years of age. The median PFS for 1st l-R, Salv-R and ASCT-R/R resp. was 0.3, 0.26 and 0.35 years resp. and 5y survival 22%, 0% and 6% resp. The median OS for 1st l-R, Salv-R and ASCT-R/R resp. was 0.55, 0.39 and 0.65 y resp. and 5y survival was 28%, 0% and 13% resp.Our results demonstrate that resistant DLBCL has an extremely poor prognosis, clearly new effective therapeutic strategies such as CAR-based therapies or others are required.This work was supported by a research program Progres Q28-9. DisclosuresBelada:Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Selectively Targeting Checkpoint Kinase By Prexasertib Had Potent Anti-Tumor Activity As Single Agent or Combined with Other Chemotherapeutic Drugs in Diffuse Large B-Cell Lymphoma, Especially Double Hit Lymphoma

Background: Relapsed/Refractory diffuse large B-cell lymphoma (DLBCL) patients had a poor prognosis, especially those relapsing within 12 months of completion therapy. Clinically, there is a need to identify and target pathways associated with acquired treatment resistance. Using rituximab-chemotherapy resistant DLBCL cell lines generated in our laboratory, we demonstrated the acquirement of rituximab resistance is associated with changes in the cell cycle distribution (S phase arrest) and expression of cell cycle regulatory proteins (cyclin B, CDC2, CDK7, Wee1, checkpoint kinase 1 [CHK1] and checkpoint kinase 2 [CHK2]). CHK1 and 2 are serine/threonine kinases that control: 1) G2/M phase transition in response of DNA damage, 2) stabilization of DNA replication fork and 3) coordinate the mitosis. Inhibition of CHKs is an attractive strategy in cancer medicine. CHK1 inhibitors are been studied in phase I clinical trials in solid tumor malignancies (i.e. breast, pancreas or lung cancer). We hypothesized that CHK inhibition by Prexasertib has effective anti-tumor activity in DLBCL pre-clinical models. Methods: We used a panel of DLBCL cell lines including rituximab sensitive (Raji, RL, DHL4, DHL6, TMD8, RIVA, and U2932), rituximab resistant (Raji 4RH and RL 4RH), and double hit DLBCL (DOH2, ROS50 and VAL). In addition, experiments were conducted in primary tumor cells isolated from lymphoma patients. Cells were exposed to prexasertib alone or in combination with chemotherapy agents. Cell viability were determined by Presto Blue assay. IC50 was calculated by Graphpad software and coefficient of synergy was calculated using the CalcuSyn software. Subsequently, cells were expose to prexasertib and changes in cell cycle distribution and apoptosis were determined by propidium iodide staining (Pi), annexing V/PI staining and by Caspase3/7 activity detection assay. Changes in mitochondrial potential were determined by DiOC6 staining. Induction of DNA double strain break was investigated by western blot probed with phosphory-H2A.X antibody. At molecular basis, downstream pathway activities ( Mcl-1, phosphor-GSK3β, phosphory-H3, and wee1) were determined by western blot. Results: Prexasertib exhibited a dose dependent anti-lymphoma activity in all all cell lines tested including DHL cell lines. The IC50 of the cells ranged from 11nM to 2.8uM at 72 hours. Additive/synergistic effects were observed by combining prexasertib and doxorubicin, etoposide, vincristine, gemcitabine and proteasome inhibitors. At the dosage of 10nM and 25nM, prexasertib induced cell cycle arrest at G2/M phase. It also induced low membrane potential, caspse3/7 activation and apoptosis in all DLBCL cell lines. Interestingly, western blot also showed CHK inhibitor at 10nM was able to reduce Mcl-1 and p-GSK 3beta. Conclusion: Our data suggests that targeting checkpoint kinase is a promising therapeutic strategy in the lymphoma, including in double hit lymphoma pre-clinical models. In vivo studies are planned. Our data supports the clinical development of prexasertib in DLBCL. (Supported by Roswell Park Cancer Institute Alliance Foundation Grant) DisclosuresNo relevant conflicts of interest to declare.

Abstract 797: Targeting HDAC3 reactivates immunosurveillance and enhances immunocheckpoint activity in B-cell lymphoma

Abstract Somatic mutation of the histone acetyltransferase CREBBP occurs in ~40% of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) patients. CREBBP mutation leads to transcriptional repression of genes involved in B-cell differentiation, antigen presentation and cell growth, and this repressive effect is mediated specifically through HDAC3. Hence CREBBP mutant DLBCL cells are biologically dependent on HDAC3 for both cell autonomous effects on cell proliferation and evasion of immune surveillance. Because of this we hypothesize that CREBBP mutant DLBCL would be highly responsive to HDAC3 specific inhibition.Therefore we treated a panel of DLBCL cell lines with increasing dose of the clinically relevant HDAC3 selective inhibitor (HDAC3i) KDAC0001 (from 0.1 to 20 μM), which resulted in a dose- and time-dependent growth inhibition and cell death. HDAC3i treatment demonstrated a stronger efficacy in cells carrying CREBBP mutation compared to the wild type with an IC50 20-30 fold lower at 72 Hrs. Knockdown by shRNA or loss of function mutations introduced via CRISPR in CREBBP wt cell lines (MD901, OCI-LY18 and RL) led to increased sensitivity to KDAC0001 compared to the parental lines. Along these lines we placed six primary DLBCL specimens (3 GCB and 3 ABC) in our 3D organoid culture system and observed potent growth suppression by KDAC001 vs vehicle after 6 days. To identify the genes through which HDAC3 drives the lymphoma phenotype we performed RNA Seq analysis using 3 DLBCL cell lines (OCI-LY1 CREBBP wt, OCI-LY19 and OZ CREBBP m) treated with either DMSO or KDAC0001 at 10 μM for 24 Hrs (a timepoint prior to cells manifesting any response to drug). We observed induction of over 1100 genes whereas 74 genes were downregulated. Gene pathway analysis showed that HDAC3 induced genes corresponded to those that are repressed in CREBBP mutant lymphomas, and regulated by BCL6-HDAC3 complexes linked to gene enhancers, including their known downstream p53 and NFKB target genes. On the other hand KDAC0001 treatment also broadly induced MHC class II and antigen presentation genes. We confirmed the increase in MHC class II genes, CIITA, PD-1 and PD-L1 not only at the transcriptional level, as determined by qPCR, but also at the protein level, as determined by flow cytometry. Co-culturing OCI-LY18 DLBCL cells and human lymphocytes with increasing amounts of HDAC3i was associated with increased cytotoxicity in a time-dependent manner. We engrafted three CREBBP wild type primary human PDX in NSG mice and treated with either vehicle or KDAC0001 at 25 or 50 mh/kg daily for 21 days. Even thought these PDX were not CREBBP mutant, there was still cell autonomous activity against all these tumors. In conclusion, HDAC3 inhibitor KDAC0001 has both cell autonomous and immune related activities.Therefore, it can be used as a novel form of “epigenetic immunotherapy” to restore immune clearance of resistant DLBCL and FL. Citation Format: Patrizia Mondello, Matt Teater, Lorena Fontan, Matthew Durant, Elisa de Stanchina, Giorgio Inghirami, Michael Green, Ari Melnick. Targeting HDAC3 reactivates immunosurveillance and enhances immunocheckpoint activity in B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 797.

Mechanisms of resistance to EZH2 inhibitors in diffuse large B-cell lymphomas

AbstractResistance to targeted therapies has become increasingly prevalent. We noted that resistance to different targeted therapies occurs by largely common mechanisms. In this study, we used this information for identifying the mechanisms of resistance to enhancer of zeste homolog 2 (EZH2) inhibitors in diffuse large B-cell lymphoma (DLBCL) harboring EZH2 mutations. We discovered that EZH2 inhibitor-resistant DLBCL cells showed activation of the insulin-like growth factor 1 receptor (IGF-1R), MEK, and phosphoinositide-3-kinase (PI3K) pathways. Constitutive activation of IGF-1R, MEK, or PI3K pathways was sufficient to confer resistance to EZH2 inhibitors in DLBCL. The activation of the PI3K/AKT and MAPK pathways decreased TNFSF10 and BAD expression through a FOXO3-dependent mechanism, which was required for the antitumor effects of EZH2i GSK126. We also identified multiple acquired mutations in EZH2 inhibitor-resistant DLBCL cell lines. These mutations independently conferred resistance to EZH2 inhibitors. Mechanistically, cellular thermal shift assays revealed that the acquired EZH2 mutations that confer resistance to EZH2 inhibitors prevent EZH2 inhibitor binding to the EZH2 mutants. Notably, EZH2 inhibitor GSK126- and EPZ-6438–resistant DLBCL cells remained sensitive to the EZH2 inhibitor UNC1999 and embryonic ectoderm development protein inhibitor EED226, which provides an opportunity to treat DLBCLs that are resistant to these drugs. Collectively, our results underpin the importance for developing a unified approach for forestalling drug resistance by prospectively considering lessons learned from the use of different targeted therapeutic agents.

Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled by Functional Lipoprotein Nanoparticles: A Therapeutic Strategy Dependent on Cell of Origin.

Cancer cells have altered metabolism and, in some cases, an increased demand for cholesterol. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative approach. Toward this end, we focused on diffuse large B-cell lymphoma (DLBCL) as a model because there is differential cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol uptake through the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). As we previously reported, GC DLBCL are exquisitely sensitive to HDL NP as monotherapy, while ABC DLBCL are less sensitive. Herein, we report that enhanced BCR signaling and resultant de novo cholesterol synthesis in ABC DLBCL drastically reduces the ability of HDL NPs to reduce cellular cholesterol and induce cell death. Therefore, we combined HDL NP with the BCR signaling inhibitor ibrutinib and the SYK inhibitor R406. By targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis, we achieved cellular cholesterol reduction and induced apoptosis in otherwise resistant ABC DLBCL cell lines. These results in lymphoma demonstrate that reduction of cellular cholesterol is a powerful mechanism to induce apoptosis. Cells rich in cholesterol require HDL NP therapy to reduce uptake and molecularly targeted agents that inhibit upstream pathways that stimulate de novo cholesterol synthesis, thus, providing a new paradigm for rationally targeting cholesterol metabolism as therapy for cancer.

Abstract 2366: Targeting PI3 pathway in ibrutinib resistant diffuse large B-cell lymphoma

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) and approximately 30% of patients with DLBCL develop relapsed/refractory disease that becomes a major cause of mortality and morbidity. The Bruton kinase (BTK) inhibitor ibrutinib successfully blocks B-cell receptor signaling and shows clinical benefit in leukemia and lymphomas, including mantle cell lymphoma (MCL) and DLBCL. Ibrutinib elicits overall desirable response rate with relapsed/refractory MCL and ABC DLBCL. However, in spite of these encouraging results, responses are variable and generally incomplete, acquired resistance is common, and recurrence and persistence of lymphoma is a problem. We undertook a study of factors underlying acquired ibrutinib resistance (IR) in initially ibrutinib-sensitive DLBCL cell lines. In this study, IR DLBCL cell lines were generated by continuous culturing of parental (PT) cell lines in increasing concentrations of ibrutinib. Once established, IR cell lines were removed from ibrutinib, expanded, and cultured under the same conditions as the PT cell lines for further experiments. Of 5 ABC DLBCL cell lines tested, two (OCI-LY3, U2932) were initially resistant to ibrutinib (IC50>10µM).Three (TMD8, OCI-LY10, HBL1) were sensitive (IC50 < 10 nM). In comparison to PT versions of these cell lines, IR cells did not form clumps in suspension cultures, displayed irregular cell morphology, showed elevated colony formation ability in methylcellulose matrix, and had a higher proliferation rate. Western blots and gene expression profile analysis showed increased expression by IR cell lines of inhibitors of apoptosis (IAP) family members, survivin, cIAP2, and oncogenic BCL2 and BCL6. The DNA damage repair pathway was found to be elevated with enhanced expression of CHK1 kinase. IR cell lines had reduced BTK expression without acquiring mutations, and downstream enhanced PI3K-Akt signaling. Analysis of PI3K isoforms revealed up-regulation of PI3Kα and PI3Kβ with decreased expression of PI3Kδ and PTEN (PI3K negative regulator). Given the enhanced PI3K isoform expression with IR, we treated IR cell lines with the PI3Kβ/δ isoform-targeting drug KA2237 and observed reduced metabolic activity (survival) of PT cell lines and further reduction of surviving IR cell lines. In conclusion, this study highlights how changes in a regulator (PTEN) and mediator (p110β) of PI3K/AKT signaling have important roles in the development of ibrutinib resistance in DLBCL. Treatment with KA2237 may provide a better outcome for ibrutinib-resistant DLBCL. Note: This abstract was not presented at the meeting. Citation Format: Neeraj Jain, Lalit Sehgal, Stephen Joseph Shuttleworth, Felipe Samaniego. Targeting PI3 pathway in ibrutinib resistant diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2366. doi:10.1158/1538-7445.AM2017-2366

Award Winner in the Young Investigator Category, 2017 Society for Biomaterials Annual Meeting and Exposition, Minneapolis, MN, April 05-08, 2017: Lymph node stiffness-mimicking hydrogels regulate human B-cell lymphoma growth and cell surface receptor expression in a molecular subtype-specific manner.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma, with multiple molecular subtypes. The activated B-cell-like DLBCL subtype accounts for roughly one-third of all the cases and has an inferior prognosis. There is a need to develop better class of therapeutics that could target molecular pathways in resistant DLBCLs; however, this requires DLBCLs to be studied in representative tumor microenvironments. The pathogenesis and progression of lymphoma has been mostly studied from the point of view of genetic alterations and intracellular pathway dysregulation. By comparison, the importance of lymphoma microenvironment in which these malignant cells arise and reside has not been studied in as much detail. We have recently elucidated the role of integrin signaling in lymphomas and demonstrated that inhibition of integrin-ligand interactions abrogated the proliferation of malignant cells in vitro and in patient-derived xenograft. Here we demonstrate the role of lymph node tissue stiffness on DLBCL in a B-cell molecular subtype specific manner. We engineered tunable bioartificial hydrogels that mimicked the stiffness of healthy and neoplastic lymph nodes of a transgenic mouse model and primary human lymphoma tumors. Our results demonstrate that molecularly diverse DLBCLs grow differentially in soft and high stiffness microenvironments, which further modulates the integrin and B-cell receptor expression level as well as response to therapeutics. We anticipate that our findings will be broadly useful to study lymphoma biology and discover new class of therapeutics that target B-cell tumors in physical environments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1833-1844, 2017.