Whole Transcriptome Sequencing Reveals Recurrent NOTCH1 Mutations in Mantle Cell Lymphoma

Abstract Abstract 436 Background: Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma that is characterized by the hallmark t(11;14)(q13;q32) translocation, as well as a high number of secondary chromosomal alterations. Further, a small number of genes such as TP53, ATM and CCND1 have been reported to be recurrently mutated in MCL, but do not fully explain the biology and do not adequately account for the wide spectrum of clinical manifestations, response to treatment and prognosis. The aim of this study was to discover new somatic mutations that could contribute to our understanding of the pathogenesis of MCL. Methods: In our discovery cohort, we sequenced the transcriptomes of 18 clinical samples (11 diagnostic and 7 progression biopsies) and 2 mantle cell lymphoma-derived cell lines (Mino and Jeko-1). For this purpose, whole transcriptome shotgun sequencing was performed on RNA extracted from fresh frozen tissue. We assembled an extension cohort of 103 diagnostic patient samples and 4 additional cell lines (Rec-1, Z-138, Maver-1, JVM-2), and performed Sanger sequencing of NOTCH1 exons 26, 27 and 34 on genomic DNA. We further exposed the 6 cell lines to 1 μM of the γ-secretase inhibitor XXI (compound E) for 7 days and measured cellular proliferation with an EdU incorporation assay. Survival analysis was carried out in the 113 patients with diagnostic biopsies and available outcome data. Results: NOTCH1 mutations were found in 14 out of 121 patient samples (11.6%) and in 2 out of 6 cell lines, Mino and Rec-1 (33.3%). The majority of these mutations (12 out of 14) lie in exon 34 that encodes the PEST domain of NOTCH1 and consist of either small frameshift-causing indels (10 cases) or nonsense mutations (2 cases). These mutations are predicted to cause truncations of the C-terminal PEST domain. To gain further insight into functional relevance, we treated 6 cell lines with compound E, an inhibitor of the γ-secretase complex that plays a critical role in the release of the intracellular domain of NOTCH1 after ligand-induced activation. In Rec-1, that harbours a NOTCH1 mutation, we observed a significant decrease in proliferation (mean percentage of cells in culture incorporating EdU decreasing from 47.5% to 1.4%, p<.001). No effect of compound E was observed in Mino, the other cell line with a NOTCH1 mutation, nor in the 4 cell lines that are wild type for NOTCH1. Outcome correlation analysis showed that NOTCH1 mutations are associated with poor overall survival (1.56 versus 3.86 years respectively, p=.001), but not with significantly shortened progression-free survival (0.88 versus 1.73 years respectively, p=.07). Discussion: We have identified recurrent mutations in NOTCH1 in a subset of patients with MCL (11.6%). The frequency and the pattern of mutations are strikingly similar to what has recently been reported in chronic lymphocytic leukemia, the other major CD5 positive B-cell malignancy (Nature, 2011 Jun 5, 475:101–105 and J Exp Med, 2011 Jul 4, 208:1389–1401). NOTCH1 mutations are associated with adverse prognosis as evidenced by shortened overall survival. This latter finding, however, should ideally be validated in a larger and uniformly treated cohort. Finally, the sensitivity of the Rec-1 cell line to compound E suggests that NOTCH1 mutations could serve as the target for tailored therapy in mantle cell lymphoma. Disclosures: Sehn: Roche/Genentech: Consultancy, Honoraria, Research Funding. Connors:Roche: Research Funding.

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Whole transcriptome sequencing reveals recurrent NOTCH1 mutations in mantle cell lymphoma

Blood ◽

10.1182/blood-2011-11-391474 ◽

2012 ◽

Vol 119 (9) ◽

pp. 1963-1971 ◽

Cited By ~ 222

Author(s):

Robert Kridel ◽

Barbara Meissner ◽

Sanja Rogic ◽

Merrill Boyle ◽

Adele Telenius ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Lines ◽

Transcriptome Sequencing ◽

Cell Lymphoma ◽

Notch Pathway ◽

Clinical Samples ◽

Mantle Cell ◽

Whole Transcriptome Sequencing ◽

Whole Transcriptome ◽

Notch1 Mutations

Abstract Mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma, is characterized by the hallmark translocation t(11;14)(q13;q32) and the resulting overexpression of cyclin D1 (CCND1). Our current knowledge of this disease encompasses frequent secondary cytogenetic aberrations and the recurrent mutation of a handful of genes, such as TP53, ATM, and CCND1. However, these findings insufficiently explain the biologic underpinnings of MCL. Here, we performed whole transcriptome sequencing on a discovery cohort of 18 primary tissue MCL samples and 2 cell lines. We found recurrent mutations in NOTCH1, a finding that we confirmed in an extension cohort of 108 clinical samples and 8 cell lines. In total, 12% of clinical samples and 20% of cell lines harbored somatic NOTCH1 coding sequence mutations that clustered in the PEST domain and predominantly consisted of truncating mutations or small frame-shifting indels. NOTCH1 mutations were associated with poor overall survival (P = .003). Furthermore, we showed that inhibition of the NOTCH pathway reduced proliferation and induced apoptosis in 2 MCL cell lines. In summary, we have identified recurrent NOTCH1 mutations that provide the preclinical rationale for therapeutic inhibition of the NOTCH pathway in a subset of patients with MCL.

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Foxo 3a Signaling Mediates Apoptosis in Lymphoma Cells By the Diterpenoid Lactone Andrographolide (Andro), the Active Component of Andrographis Paniculata

Blood ◽

10.1182/blood.v126.23.2760.2760 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2760-2760

Author(s):

Shuo Yang ◽

Bo Ding ◽

Fei Ying ◽

Jana Svetlichnaya ◽

Austin Tom ◽

...

Keyword(s):

Cell Cycle ◽

Mantle Cell Lymphoma ◽

Signaling Pathways ◽

Cell Line ◽

Cell Lines ◽

Cell Lymphoma ◽

Wild Type ◽

Mantle Cell ◽

Golgi Fragmentation ◽

Bcl2 Expression

Abstract Introduction: Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an herbal medicine used in Asia. It has been reported to have anti-inflammatory, antihypertensive, antiviral, and immune-stimulant properties. Furthermore, it has been shown to inhibit cancer cell proliferation and induce apoptosis in lymphoma, leukemia and other solid tumor cell lines. We have shown that Andro caused ROS-dependent apoptosis in lymphoma cell lines and in primary tumor samples that was mediated through mitochondrial pathways and enhanced by depletion of GSH and inhibited by NAC or the pan-caspase inhibitor Z-VAD-FMK (Yang et al Clin Cancer Res 2010; 16(19):4755). We hypothesized that the tumor suppressor, FOXO3a may be involved in signaling pathways that lead to apoptosis and to test that hypothesis we investigated the role of FOXO3A in Andro induced signaling in lymphoma. Methods: We studied the Burkitt p53-mutated Ramos cell line, the mantle cell lymphoma (MCL) line Granta, the transformed follicular lymphoma (FL) cell line HF-1, and the diffuse large B-cell lymphoma (DLBCL) cell line SUDHL4, as well as primary cells from patients with FL and MCL. We transfected shRNA FOXO3a by electroporation to build stable cells with constant knockdown of FOXO3a in Ramos and SUDHL4 cell lines. We then compared the cell viability (MTT and Golgi fragmentation), apoptosis (Annexin V by flow), c-MYC and Bcl2 expression, death receptors 4 (DR4) expression and cell cycle related proteins in wild type and FOXO3a knockdowns. Results: We found that Andro resulted in nuclear translocation of FOXO3a in Ramos at early time points. We found that shRNA stable knockdown of FOXO3a in Ramos and SUDHL4 cell lines protected cells (Ramos and SUDHL4) from Andro-induced apoptosis (Figure 1). Moreover, in multiple cell lines, we found that Andro decreased c-MYC expression, which was abrogated in part by FOXO3A knockdown compared with wild type cells. Similarly, reduction in mitochondrial membrane potential by Andro is abrogated in the FOXO 3a knockdown cells. These data suggest that FOXO3a regulates c-MYC stabilization by mitochondrial proteins (for example TFAM and MAD-1). In the Granta cell line, derived from Mantle Cell Lymphoma (MCL) and in an MCL patient sample, Andro reduced c-MYC expression. We also found that Andro induced Death Receptor 4 (DR4) at the mRNA and protein level in Granta cells in a dose-dependent manner. The cell cycle control proteins Aurora, p21, p27 (the latter 2 regulated by FOXO3a), are also increased by Andro. When cell death was measured by Golgi fragmentation and subsequent collapse, we found that Andro induced Golgi fragmentation in Granta and SUDHL4 cells Conclusion: Andro-induced lymphoma cell apoptosis is mediated through multiple signaling pathways, including FOXO3a, which appears to play a significant role, perhaps by regulating c-MYC stabilization and BCL2 expression and cell cycle proteins. These data suggest that this novel diterpenoid lactone compound deserves further pre-clinical and clinical testing in malignant lymphoma. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Genome-Wide Analysis of BRD4-Targets Reveals the Therapeutic Relevance of Simultaneous Targeting of BCR Pathway and IKZF-MYC Axis in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2018-99-109982 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4100-4100

Author(s):

Junya Kuroda ◽

Taku Tsukamoto ◽

Shingo Nakahata ◽

Kazuhiro Morishita ◽

Ryuichi Sato ◽

...

Keyword(s):

Cell Proliferation ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Research Funding ◽

Target Genes ◽

Cell Lymphoma ◽

Genome Wide ◽

Mantle Cell ◽

Bcr Signaling ◽

Dose Dependent

Abstract Mantle cell lymphoma (MCL) has been mostly incurable, and there is an urgent need to identify targetable molecules for development of a more effective treatment strategy. Bromodomain and extraterminal domain (BET) proteins associate with acetylated histones and facilitate transcription of target genes, and bromodomain-containing 4 (BRD4), a member of BET proteins, recruits the P-TEFb complex to genomic lesions in chromatin and thereby activates RNA Pol II at specific promoter sites of target genes. In addition, super-enhancers have been recognized as regulatory regions with a high level of acetylated histones, mediator complexes and BRD4, and super-enhancers in cancer cells are enriched at oncogenes. Recent studies have shown that BRD4 promotes expression of pivotal molecules in disease development, maintenance and progression in various cancers, including lymphoma. Given, we in this study examined the effect of BRD4 inhibition on human MCL-derived cell lines, Jeko-1, JVM-2, MINO and Z138, and performed broad screening of BRD4-regulated molecules using genome-wide approaches to identify therapeutic targets for MCL. As the results, treatment with a BRD4 inhibitor I-BET151 for 72 h showed a dose-dependent inhibitory effect on cell proliferation in all four cell lines, with half maximal inhibitory concentrations (IC50s) of 15.6 nM, 3.6 nM, 2.6 nM and 3.0 nM in Jeko-1 cells, JVM2 cells, MINO cells and Z138 cells, respectively, which was accompanied by G1/S cell cycle arrest and the induction of apoptosis. Next, we performed comprehensive gene expression profile (GEP) analysis for JVM2 and Z138 cells with or without I-BET151 treatment, and BRD4 chromatin immunoprecipitation sequencing (ChIP-Seq) in JVM2 cells treated with 10 nM I-BET151 or DMSO. Accordingly, GEP analyses revealed that more than 600 genes were commonly upregulated by more than 1.5-fold and downregulated by less than 0.67-fold, respectively, in JVM2 and Z138 cells treated by I-BET151, while ChIP-Seq showed that 7988 BRD4-binding regions were dysregulated by I-BET151, with most of these sites in enhancer regions, and 547 BRD4-binding regions were characterized as super-enhancers. Integrated analysis using the Reactome Pathway Database and the results of GEP and ChIP-Seq showed that a series of genes involved in the B cell receptor (BCR) signaling pathway and IKZF-MYC axis are regulated by BRD4 in MCL cells. To confirm whether each BRD4 target contributes to survival and proliferation of MCL cells, we focused on several candidate targets: the BCR pathway, IKZF and MYB. However, ibrutinib, a Bruton kinase inhibitor, suppressed cell growth in only two of the four cell lines (MINO and JVM2) in a dose-dependent manner, while lenalidomide, an inhibitor of the IKZF family, did not affect cell survival, despite its potency in decreasing IKZF1 and IKZF3 proteins. MYB silencing using shMYB did not decrease cell proliferation in any of the four MCL cell lines. In conclusion, our study disclosed that BRD4 regulates transcription of multiple genes by binding to enhancer region, partly involving super-enhancers and multiple known pathways, such as BCR signaling and the IKZF-MYC axis, which play essential roles in survival of MCL cells. While the efficacy of single targeting of BCR-signaling, IKZF, or MYB was limited, I-BET151 concomitantly inactivated the BCR pathway and IKZF and had a high growth inhibitory efficacy in MCL cells. These results suggest that simultaneous targeting of multiple molecules involved in the BCR pathway and IKZF-MYC axis may overcome resistance to ibrutinib and/or lenalidomide in MCL, and that BRD4 inhibitors are promising candidates for MCL treatment. Disclosures Kuroda: Chugai Pharma: Honoraria, Research Funding. Taniwaki:Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.,: Research Funding; Astellas Pharma Inc,: Research Funding.

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In Vitro and In Vivo Anti Lymphoma Effect of GX15-070 in Mantle Cell Lymphoma.

Blood ◽

10.1182/blood.v108.11.4756.4756 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4756-4756 ◽

Cited By ~ 1

Author(s):

Gwyn Bebb ◽

Huong Muzik ◽

Sophia Nguyen ◽

Don Morris ◽

Douglas A. Stewart

Keyword(s):

Mantle Cell Lymphoma ◽

Cell Line ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cytotoxic Agents ◽

In Vivo Experiments ◽

Mantle Cell

Abstract Introduction Mantle cell lymphoma (MCL), an incurable B cell lymphoma, consistently over expresses bcl-2 despite not carrying the t(14;18). The attenuation of apoptosis by bcl-2 is thought to contribute to the malignant process and increase resistance to some cytotoxic agents. We recently demonstrated that GX15-070, a small molecular inhibitor of the BH3 binding groove of bcl-2, has activity against MCL cell lines in vitro. We set out to assess the effect of GX15-070 alone and in combination with Vincristine on the viability of MCL cells in vitro and in vivo. Methods 3 previously characterized bcl-2 over expressing MCL cell lines (JVM-2, Hbl-2, granta) were used. Cells were grown in standard media and exposed to a range of concentrations of GX15-070 with and without Vincristine. Dose-response was assessed by measuring viability at 48 hours using the WST-1 assay. In vivo experiments were conducted on immune deficient mice in which 5×106 cells were injected in the flank then treated IV with GX15-070 (q 2days × 5 doses), Vincristine (q4 days × 3 doses) or both starting 5 days later. Tumours were measured three times weekly. Results All three MCL cell lines over-expressed bcl-2 by western blot. Each MCL cell line showed sensitivity to GX15-070 at a range of concentrations. The addition of GX15-070 to low dose Vincristine (10−6) caused significant growth inhibition of each MCL cell line (see table 1). Discussion Our results demonstrate that using GX15-070 to target bcl-2 is an effective anti neoplastic approach against MCL cell lines in vitro. In addition, our results suggest that combining Vincristine and GX15-070 is a promising strategy in treating MCL. In vivo experiments to confirm this additive activity are still ongoing and will be presented in full. Initial impressions suggest that there is a rationale for the addition of GX15-070 to current cytotoxic regimens used to treat MCL in the setting of clinical trials. Table 1: Effect of Vincristine and GX15-070 on in vitro growth of 3 MCL cell lines Growth as % age of Control Cell Line JVM-2 HBL-2 Granta Vincristine alone (10-6 mg/ml) 92% 48% 89% GX15-070 alone (0.08 uM) 75% 76% 60% Vincristine 10-6 mg/ml and GX15-070 0.08 uM 52% 24% 52%

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Anti-CD20 Antibody GA101 Shows Higher Cytotoxicity but Is Competitively Displaced by Rituximab in Mantle Cell Lymphoma.

Blood ◽

10.1182/blood.v114.22.2704.2704 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2704-2704

Author(s):

Daniel A. Heinrich ◽

Christian Klein ◽

Kristina Decheva ◽

Marc Weinkauf ◽

Grit Hutter ◽

...

Keyword(s):

Western Blot ◽

Cell Viability ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

Deutschland Gmbh ◽

Mantle Cell ◽

Cd20 Antibody ◽

Anti Cd20

Abstract Abstract 2704 Poster Board II-680 Background: Mantle cell lymphoma (MCL) is characterized by a poor long-term prognosis with a median survival of 3–5 years. Type I anti-CD20 antibody rituximab has demonstrated a clear anti-proliferative effect in MCL and achieves increased response rates in combination with chemotherapy. GA101, a third-generation IgG1 anti-CD20 antibody displays improved ADCC and superior direct cell death induction by virtue of glycoengineering compared to rituximab and its targeting a type II epitope on CD20, respectively. Methods: Using a panel of MCL cell lines (Rec-1, HBL-2, Jeko-1, Granta-519, JVM-2 and Z-138) we determined the effect of GA101 alone as well as in combination with rituximab on cell viability and proliferation. Karpas-422 (Diffuse Large B-Cell Lymphoma) was used as a control cell line. MCL and Karpas-422 cells were treated with GA101 or rituximab at concentrations of 1 – 20μg/ml and rituximab. Cell viability was analyzed by trypan-blue exclusion tests at 0h, 24h, 48h and 72h. The panel of MCL cell lines and Karpas-422 were then treated with GA101 and rituximab each at 1 and 10 μg/ml to determine potential synergism of antibody combinations. Accordingly, a fractional product calculation was performed: synergism > 0,1; antagonism < −0,1. In addition, Western-blot and RNA-array-analyses were performed to elucidate potential intra-cellular downstream pathway mechanisms. Results: After mono-exposure with GA101 (1 μg/ml), Granta-519 and Rec-1 showed the highest sensitivity (65–75% cell reduction in Granta-519 and 35–40% in Rec-1). Intermediate results were gained for Z-138, HBL-2, Jeko-1 and JVM-2 and Karpas-422 (15–20%). rituximab mono-exposure at 12,5 μg/ml showed a 25% reduction of cell count in Granta-519, 20% in HBL-2 and < 5% in Rec-1, Jeko-1 and Z-138. Combination experiments suggested the competitive binding of the two antibodies. Thus, GA101 plus rituximab combination experiments resulted in a lower cytotoxicity than GA101 alone, according to fractional product calculations. Conclusions: Although GA101 is competitively displaced by rituximab, GA101 demonstrates higher efficacy in MCL cell lines than rituximab, even at a more than 10-fold lower concentration. Currently RNA-array- and Western blot analysis are being performed to identify the critical pathways responsible for the superior cytotoxicity of GA101. Disclosures: Klein: Discovery Oncology, Roche Diagnostics GmbH: Employment. Weinkauf:Lilly Deutschland GmbH: Research Funding. Hutter:Lilly Deutschland GmbH: Research Funding. Zimmermann:Lilly Deutschland GmbH: Research Funding. Dreyling:Roche: Honoraria, Research Funding.

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Deferred Treatment Is Associated with Improved Overall Survival in Patients with Newly Diagnosed Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v126.23.2717.2717 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2717-2717

Author(s):

Jonathon B. Cohen ◽

Xuesong Han ◽

Xin Hu ◽

Ahmedin Jemal ◽

Elizabeth Ward ◽

...

Keyword(s):

Overall Survival ◽

Mantle Cell Lymphoma ◽

Research Funding ◽

Cell Lymphoma ◽

Early Stage ◽

Cohort Analysis ◽

Newly Diagnosed ◽

Early Stage Disease ◽

Deferred Treatment ◽

Mantle Cell

Abstract Background: Although mantle cell lymphoma (MCL) has traditionally been considered an aggressive lymphoma with shortened survival, the long-term outcomes and initial presentation can be heterogeneous. Martin et al (JCO 2009) reported that 32% of patients with MCL at an academic referral center deferred therapy for at least 3 months, with a median overall survival (OS) of 64 months for patients treated within 90 days of diagnosis and median OS not reached for those who deferred therapy. We used the National Cancer Database (NCDB) to perform a national cohort analysis of the impact of deferred therapy in MCL. Methods: The NCDB is a nationwide oncology outcomes database sponsored by the Commission on Cancer of the American College of Surgeons and the American Cancer Society, capturing nearly 70% of all newly diagnosed cases of cancer in the United States. We included all patients ≥18 years old who received initial treatment for newly diagnosed MCL in 2004-2011. MCL patients were identified by the International Classification of Diseases for Oncology code 9673 and variables of interest were captured using the Facility Oncology Registry Data Standards. Patients were determined to have received deferred therapy if their time to initial treatment was > 90 days. Chi-square tests were used as appropriate to compare baseline characteristics between immediate and deferred treatment groups, and OS was estimated using the Kaplan-Meier method. Log-binomial regression models were developed to identify characteristics associated with deferred treatment and multivariable Cox proportional hazard models were fit to evaluate the relationship between deferred treatment and OS. Results: Of 8209 patients with MCL, 492 (6.1%) received therapy > 90 days from diagnosis with a median time to treatment for this group of 121 days (range 91-1152). Among all patients, 64% were > 60 years of age, 73% were male, 85% were stage III/IV, and 83% had primarily nodal disease. Additional comorbidities were identified in 22% of patients, and 28% of patients presented with B-symptoms at diagnosis. Approximately 1/3 of patients received therapy in a high volume teaching/research institution. Compared to patients treated within 90 days of diagnosis, patients receiving deferred therapy were more likely to have early stage disease (22% vs 15% p<0.0001), extranodal presentation (24% vs 17%, p<0.0001), to be located in the Northeast region (26% vs 20%, p<0.0001), and to be treated at a high volume teaching/research institution (41% vs 33%, p=0.005). Patients treated within 90 days of diagnosis more commonly had B-symptoms (29% vs 16%, p<0.0001). There were no significant differences between the two groups with regard to gender, age, year of diagnosis, socioeconomic status (based on location of residence), or primary payer. When analyzed in a multivariable model, lack of B-symptoms (RR 1.67, 95% CI 1.38-2.03, p < 0.0001) and extra-nodal status (RR 1.24, 95% CI 1.00-1.53, p = 0.0468) were two strong clinical predictors of deferred therapy. Multivariable analysis demonstrated improved OS for patients who received deferred therapy (HR 0.79: 95% CI 0.67-0.93, p = 0.005; See Figure 1). Additional significant predictors of improved OS included age ≤ 60 years (HR 0.60: 95% CI 0.54-0.66, p < 0.0001), early stage disease (HR 0.66: 95% CI 0.59-0.74, p < 0.0001), lack of B-symptoms (HR 0.75: 95% CI 0.70-0.81, p < 0.0001), and lack of comorbidities (HR 0.63: 95% CI 0.58-0.68, p < 0.0001). Non-Hispanic black patients had inferior OS compared to the other racial/ethnic groups. Among patients who deferred therapy, male gender (p=0.046), age ≤ 60 years (p=0.0002) and lack of comorbidities (p<0.0001) were associated with improved OS, while remaining variables including region, stage, race, B-symptoms, and extranodal presentation were not. Discussion: This national cohort analysis supports prior reports that deferred therapy in MCL is safe for a subgroup of patients with MCL. We found that deferred therapy > 90 days was associated with improved OS and that lack of B-symptoms was a strong predictor for deferred therapy. Predictors of improved survival for patients deferring therapy included young age and lack of comorbidities. These data support use of watchful waiting approach for well-selected newly diagnosed MCL patients. Figure 1. Overall survival for newly diagnosed patients with mantle cell lymphoma based on time to initiation of therapy. Figure 1. Overall survival for newly diagnosed patients with mantle cell lymphoma based on time to initiation of therapy. Disclosures Cohen: BMS: Research Funding; Seattle Genetics: Consultancy; Pharmacyclics: Consultancy; Millennium: Consultancy; Celgene: Consultancy; Janssen: Research Funding. Flowers:Infinity Pharmaceuticals: Research Funding; Acerta: Research Funding; Millennium/Takeda: Research Funding; AbbVie: Research Funding; Gilead Sciences: Research Funding; Acerta: Research Funding; Gilead Sciences: Research Funding; Onyx Pharmaceuticals: Research Funding; Janssen: Research Funding; OptumRx: Consultancy; Spectrum: Research Funding; Seattle Genetics: Consultancy; Infinity Pharmaceuticals: Research Funding; Genentech: Research Funding; Millennium/Takeda: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Onyx Pharmaceuticals: Research Funding; Pharmacyclics: Research Funding; Spectrum: Research Funding; Pharmacyclics: Research Funding; AbbVie: Research Funding; Seattle Genetics: Consultancy; Celegene: Other: Unpaid consultant, Research Funding; OptumRx: Consultancy.

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Targeting Oxphos Pathway Against Ibrutinib Resistance to Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v128.22.290.290 ◽

2016 ◽

Vol 128 (22) ◽

pp. 290-290 ◽

Cited By ~ 1

Author(s):

Yang Liu ◽

Taylor Bell ◽

Hui Zhang ◽

Yuting Sun ◽

Carrie J Li ◽

...

Keyword(s):

Tumor Growth ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

Gene Set Enrichment Analysis ◽

Pdx Model ◽

Mantle Cell ◽

Ibrutinib Resistance

Abstract Background: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy that is initially responsive but ultimately relapses to frontline therapy. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK) has achieved 68% of overall response rate in relapsed/refractory mantle cell lymphoma (MCL) patients. However, the vast majority of MCL patients experience disease progression, demonstrating that standard-of-care approaches are failing and that a means for targeting ibrutinib resistant MCL is clinically needed. Our hypothesis is that the ibrutinib-resistant MCL may rely on the mitochondrial oxidative phosphorylation (OXPHOS) pathway to produce energy for tumor growth. In this study, we investigated the effects of IACS-010759, a small molecule mitochondrial complex I inhibitor discovered in MD Anderson Cancer Center which can block the OXPHOS pathway, to overcome ibrutinib resistance in MCL in vitro and in a patient-derived xenograft (PDX) model. Methods: The OXPHOS metabolic pathways were investigated by RNASeq in a panel of ibrutinib-sensitive and -resistant MCL samples. Cell growth inhibition assays were tested after 72-hour treatment with IACS-010759 in ibrutinib-resistant MCL cell lines, Z-138 and Maver-1, and ibrutinib-sensitive MCL cell lines, Rec-1, Mino, and Jeko-1, by CellTiter-Glo luminescent cell viability assay (Promega). Furthermore, an IBN-resistant MCL PDX model was established and the therapeutic effects and tolerability of IACS-010759 were investigated in the primary MCL-bearing PDX model. Results: We have done RNA sequencing (RNASeq) in 7 primary ibrutinib-resistant and 16 ibrutinib-sensitive MCL patient samples, and analyzed the data using Gene Set Enrichment Analysis (GSEA) software. The results demonstrated that the OXPHOS pathway was activated in the primary ibrutinib-resistant MCL cells but not ibrutinib-sensitive MCL cells. Based on the RNASeq data, we selected an OXPHOS inhibitor IACS-010759 to investigate its effects on both primary ibrutinib-resistant and ibrutinib-sensitive MCL cells in vitroand in PDX mice. IACS-010759 significantly inhibited cell proliferation in ibrutinib-resistant MCL cell lines, Z-138 and Maver-1, but not in ibrutinib-sensitive MCL cell lines, Rec-1, Mino, and Jeko-1, during a 72-hour incubation. Furthermore, the primary ibrutinib-resistant MCL PDX mice were administrated with 10 mg/kg IACS-10759 by oral gavage, for 28 days using a 5 on/2 off dosing schedule. Our data showed that IACS-010759 completely eradicated tumor growth in ibrutinib-resistant MCL PDX mice (n=5, p=0.045). All mice tolerated the treatment dose and no toxicity was found during 28 days of IACS-010759 treatment. Conclusions: The OXPHOS inhibitor IACS-010759 overcomes ibrutinib resistance both in vitro and in the PDX mouse model. The investigation of its mechanism-of-action is ongoing. IACS-010759 could have the potential for clinical use in ibrutinib-resistant relapsed/refractory MCL patients. Disclosures Wang: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Asana BioSciences: Research Funding; Kite Pharma: Research Funding; Juno Therapeutics: Research Funding; Asana biosciences, Beigene, Celgene, Juno, Kite, Onyx, Pharmacyclics: Research Funding; Dava Oncology: Honoraria; BeiGene: Research Funding; Acerta: Consultancy, Research Funding.

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Venetoclax Resistance in Mantle Cell Lymphoma Is Mediated By BCL-XL and Can be Circumvent By Inhibiting the BH4 Domain of BCL-2

Blood ◽

10.1182/blood-2019-127931 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1507-1507

Author(s):

Daniela Steinbrecher ◽

Felix Seyfried ◽

Eugen Tausch ◽

Johannes Bloehdorn ◽

Billy Michael Chelliah Jebaraj ◽

...

Keyword(s):

Cell Viability ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Research Funding ◽

High Efficiency ◽

Cell Lymphoma ◽

Fold Increase ◽

Dependent Manner ◽

Independent Manner ◽

Mantle Cell

Apoptosis is controlled by the expression levels and interplay of pro- and anti-apoptotic BCL-2 family proteins. The specific BCL-2 inhibitor Venetoclax (VEN) showed high efficiency in BCL-2 dependent cancers like chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). Despite its high efficiency in CLL and MCL, refractory disease can develop. BCL-2 mutations have been described to mediate resistance in CLL cases, however these mutations are only found in a proportion of VEN resistant cases and in a fraction of cells. In order to design alternative therapeutic strategies to overcome drug resistance, a better understanding of the mechanisms mediating resistance to VEN is necessary. VEN-resistant (VEN-R) MCL cell lines (MINO and MAVER-1) were generated by chronic exposure to increasing amounts of VEN (up to 3µM). A significant and stable upregulation of BCL-XL mRNA and protein was seen in the MINO and MAVER-1 resistant cell lines (2 and 4 fold increase in mRNA and 2.6 and 4.5 fold increase in protein, respectively). We used BH3 profiling in combination with VEN treatment for 4h to investigate the differences in anti- and pro-apoptotic signaling in parental and VEN-R cell lines. Additionally, sensitivity to VEN was restored upon shRNA-mediated knockdown of BCL-XL. These results confirmed the importance of BCL-XL upregulation in mediating resistance. Furthermore, we did not detect mutations in BCL-2 upon resistance to VEN via targeted NGS, which is in contrast to results obtained in VEN-R CLL patients (Blombery et al., Cancer Discovery 2019 and Tausch et al., Hematologica 2019). However, the results obtained by dynamic BH3-profiling (VEN treatment in combination with BH3 Profiling) suggest that increase in BCL-XL is most likely not the only alteration necessary to render cells resistant to VEN. In addition, reduced activation of pro-apoptotic proteins like BAX and BAK might contribute to resistance to VEN. In order, to investigate if VEN resistance can be overcome by drug mediated inhibition of BCL-XL we used different therapeutic approaches. Combinational treatment with the BCL-XL inhibitor A-1331852 and VEN or the single treatment with Navitoclax, a combined inhibitor of BCL-2, BCL-W and BCL-XL for 48h reduced cell viability in VEN-R MINO and MAVER-1 cell lines. Furthermore, BDA-366, a BH4 domain BCL-2 inhibitor effectively reduced the cell viability after 48h of treatment in a dose dependent manner in both parental and VEN-R cell lines. The binding of BDA-366 to the anti-apoptotic BCL-2 protein leads to a conformational change into a pro-apoptotic molecule by the exposure of the BH3 domain of the protein. Despite mediating apoptosis in a TP53-independent manner, VEN treatment in CLL has been associated with inferior outcome in the presence of TP53 aberrations. In order to address the role of TP53 dysfunction in mediating resistance to VEN, we generated p53 knock out cell lines (N=2) by CRISPR/Cas9 gene editing. This significantly decreased the sensitivity to VEN compared to p53 WT cell lines. Additionally, the sensitivity to BDA-366 was significantly reduced upon knockout of p53, suggesting an interference of p53 downstream of BCL-2. Overall, VEN resistance is mediated by a permanent increase in BCL-XL mRNA and protein level in MCL. Importantly, BDA-366, which converts the anti-apoptotic BCL-2 molecule into a BAX-like death molecule, could be a potential alternative treatment strategy for BCL-2 dependent cancers even when resistant to VEN. Despite mediating apoptosis in a p53 independent manner, VEN seems to be less effective in p53 deficient cells, underlining the importance of further investigations of treatment combinations in these groups. Disclosures Tausch: Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Stilgenbauer:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau. Schneider:Celgene: Other: travel grant.

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Targeting Transcription Checkpoint Using a Novel CDK9 Inhibitor in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2020-140865 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-29

Author(s):

Junwei Lian ◽

Yu Xue ◽

Alexa A Jordan ◽

Joseph McIntosh ◽

Yang Liu ◽

...

Keyword(s):

B Cell ◽

Mantle Cell Lymphoma ◽

Mouse Models ◽

Cell Lines ◽

Cell Apoptosis ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Mantle Cell ◽

Cdk9 Inhibitor

Introduction Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma that accounts for 5-8% of all non-Hodgkin lymphomas. Despite the Bruton's tyrosine kinase inhibitor ibrutinib and the BH3 mimetic BCL2 inhibitor venetoclax (ABT-199) have proven to be effective therapeutic strategies for MCL, most patients often experience disease progression after treatment. Thus, developing a novel drug to overcome this aggressive relapsed/refractory malignancy is an urgent need. Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase belonging to the CDK family which regulates multiple cellular processes, particularly in driving and maintaining cancer cell growth. Unlike classical CDKs, CDK9 is a critical component of the positive transcription elongation factor b (P-TEFb) complex that mediates transcription elongation and mRNA maturation via phosphorylating RNA polymerase II (RNAP2). Previous studies demonstrated that CDK9 inhibition downregulates transcription levels of MCL-1 and MYC, which are crucial in both survival and proliferation of acute myeloid leukemia and diffuse large B-cell lymphoma. We and others found that the MYC signaling pathway was enhanced in MCL, especially in ibrutinib-resistant MCL patients. MYC is a core transcription factor driving lymphomagenesis. It does not possess enzymatic activity and has long been considered to be undruggable. MCL-1 is a key anti-apoptotic protein and is overexpressed in several hematologic malignancies. It was also found to be overexpressed in ibrutinib or venetoclax-resistant MCL cells. Thus, CDK9 is considered as a potential target that may inhibit MYC and MCL-1 pathways. Although recently it was shown that MC180295, a novel selective inhibitor of CDK9, has nanomolar levels anti-cancer potency, whether its beneficial effects extend to relapsed/refractory MCL has not yet been assessed. Methods We use three paired MCL cells sensitive/resistant to ibrutinib or venetoclax to test the efficacy of CDK9 inhibitor MC180295. Cell viability was measured by using Cell Titer Glo (Promega). Cell apoptosis assay and western blot analyses were used to identify affected pathways after MC180295 treatment. Finally, we used patient-derived xenograft (PDX) mouse models to test the therapeutic potential of MC180295 in MCL. Results First, we examined the potential efficacy of a CDK9 inhibitor MC180295 in MCL cells. MC180295 treatment results in growth inhibition of ibrutinib-resistant or venetoclax-resistant MCL cells. By assessing the caspase 3 and PARP activity, we found that MC180295 treatment induces cell death via cell apoptosis in MCL cell lines. Meanwhile, we found that RNAP2 phosphorylation at Ser2, the active form of RNAP2, is downregulated in MC180295 treated MCL cell lines. Consistent to previous studies, MC180295 treatment significantly reduces the protein level of MYC and MCL-1. In addition, we identified several other important proteins, such as cyclin D1 and BCL-XL, were also downregulated upon MCL180295 treatment. MC180295 was able to overcome ibrutinib-venetoclax dual resistance in PDX mouse models without severe side effects. To improve the efficacy of MC180295 as a single agent, we performed in vitro combinational drug screen with a number of FDA-approved or investigational clinical agents and found that MC180295 had a synergistic effect with venetoclax. We are currently investigating the underlying mechanism of action. Conclusion Taken together, our findings showed that targeting CDK9 by its specific inhibitor MC180295 is effective in targeting MCL cells, especially those with ibrutinib or venetoclax resistance and therefore supports the concept that CDK9 is a new target to overcome ibrutinib/venetoclax resistance in MCL. Disclosures Wang: MoreHealth: Consultancy; Dava Oncology: Honoraria; Beijing Medical Award Foundation: Honoraria; OncLive: Honoraria; Molecular Templates: Research Funding; Verastem: Research Funding; Guidepoint Global: Consultancy; Nobel Insights: Consultancy; Oncternal: Consultancy, Research Funding; InnoCare: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Lu Daopei Medical Group: Honoraria; Pulse Biosciences: Consultancy; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding.

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UBR5 Mutations in Mantle Cell Lymphoma Lead to Increased Proliferation through a Cyclin D1-Dependent Mechanism

Blood ◽

10.1182/blood-2018-99-118524 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2849-2849

Author(s):

Olga Kutovaya ◽

Stacy Hung ◽

Elena Viganò ◽

Adele Telenius ◽

Bruce W Woolcock ◽

...

Keyword(s):

Cell Cycle ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

Gene Mutations ◽

Non Hodgkin Lymphoma ◽

Expression Of Genes ◽

Mantle Cell

Abstract Mantle cell lymphoma (MCL) is an aggressive type of non-Hodgkin lymphoma, with patient outcomes inferior to most other lymphoma subtypes. Recent progress in describing recurrent somatic gene mutations has led to a better understanding of MCL pathogenesis. However, the functional and clinical implications of many alterations remain to be elucidated. Here, to uncover the role of recurrent UBR5 gene mutations in lymphomagenesis, we studied a cohort of 248 MCL patients by targeted sequencing and performed genome editing of MCL-derived cell lines to investigate UBR5-mutation associated phenotypes in vitro. We identified deleterious UBR5 exon 58 hotspot mutations in 8% of MCL patients, all of which were mutually exclusive with CCND1 mutations. Proteomics analysis of Granta-519 and Jeko-1 cell lines with engineered UBR5 exon 58 indel mutations showed differential expression of genes involved in cell cycle and ubiquitination, and led to the discovery of decreased phosphorylation of CCND1 in the UBR5-mutated lines. Accordingly, in vitro studies of engineered genome-edited Granta-519, Jeko-1 and Mino cells revealed accumulation of cells in the S phase of the cell cycle, increased phosphorylation of retinoblastoma protein (Rb), and increased lymphoma cell proliferation. Our results demonstrate that UBR5 mutations, in addition to the hallmark t(11;14) translocation drive proliferation of MCL cells, potentially rendering mutation-carrying cells more sensitive to targeted therapies. Disclosures Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Roche: Research Funding; Celgene: Consultancy, Honoraria; Janssen: Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: patent holding; Juno Therapeutics: Consultancy; Seattle Genetics: Consultancy; Tioma: Research Funding.

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