scholarly journals Targeting PRMT5 to Circumvent Acquired Ibrutinib Resistance in Mantle Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4065-4065 ◽  
Author(s):  
Shelby Sloan ◽  
Fiona Brown ◽  
JI Hyun Chung ◽  
Alexander Prouty ◽  
Esther Wheeler ◽  
...  
Keyword(s):  
Research Funding ◽  
Disease Burden ◽  
Cell Lymphoma ◽  
Ex Vivo ◽  
Ohio State University ◽  
State University ◽  
University Patents ◽  
Mantle Cell ◽  
Ibrutinib Resistance ◽  
Dimethyl Arginine

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by genetic dysregulation of cyclin D1 and activation of signaling pathways driving uncontrolled MCL cell proliferation and survival. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton's tyrosine kinase (BTK), a downstream target of the B-cell receptor (BCR) pathway. While ibrutinib exhibits significant single-agent therapeutic activity in patients with relapsed/refractory MCL, the vast majority of MCL patients on ibrutinib progress with aggressive disease and short survival (3-8 mo). Although ~80% of chronic lymphocytic leukemia patients with acquired ibrutinib resistance have mutations in BTK and PLCγ2, this is uncommon in MCL suggesting alternative mechanisms driving this resistant phenotype. Understanding drug-resistance mechanisms and developing effective therapies for ibrutinib resistant (IR) MCL are urgently needed. The major type II protein arginine methyltransferase enzyme, PRMT5, catalyzes symmetric dimethylation of arginine residues on histone tails (H3R8 and H4R3) and other proteins. PRMT5 regulates a vast array of biologic functions including RNA processing, DNA damage response, signal transduction, and gene expression. Amplified PRMT5 activity drives the expression and activity of key oncogenes (MYC, CYCLIND1, NOTCH1) while silencing expression and activity of tumor suppressors (ST7, RBL2, and p53). Our group has shown PRMT5 is overexpressed and dysregulated in MCL and strategies aimed at selectively targeting PRMT5 show anti-tumor activity in preclinical lymphoma models. Here we describe the development of a novel patient derived xenograft (PDX) of IR-MCL and explore PRMT5 inhibition as an alternative therapeutic option to circumvent IR. Peripheral blood mononuclear cells from a 75 yo male patient diagnosed with acquired classic IR-MCL were engrafted intravenously into NSG mice. After 5 passages, all mice engrafted with 107 MCL cells developed histologically confirmed MCL infiltrating kidney, lymph nodes, bone marrow, spleen and peripheral blood. Circulating human CD5+/CD19+ cells were detectable and quantifiable by flow cytometry by day 21 post-engraftment. Karyotype analysis confirmed the hallmark t(11;14)(q13;q32) of MCL while retaining nearly all cytogenetic abnormalities present in the patient's primary tumor including a deletion of chromosome 9, associated with deletion of MTAP, a therapeutic vulnerability for PRMT5-targeted therapy. Whole exome sequencing confirmed genomic stability with successive passages. Ex vivo cytotoxicity assays and protein pathway analysis further confirmed resistance to ibrutinib (IC50 >1 µM) with maintained hyper-phosphorylation of AKT (Ser473) and ERK (Thr202/Tyr204). Western blot analysis showed elevated levels of c-MYC, CYCLIND1, BCL2, and pERK. After validation of circulating disease at day 25 post engraftment, mice were treated with either a novel small molecule inhibitor of PRMT5 (PRT382, 10 mg/kg orally 4 days on 3 days off) or ibrutinib (75 mg/kg administered in drinking water, n=5 mice per treatment group). Treatment of this PDX model with PRT382 resulted in significantly decreased disease burden and improved median survival compared to control animals from 48 to 83 days, respectively (p=0.0045). We found no significant difference in survival (p= 0.6540) or circulating disease burden with ibrutinib therapy compared to control group. The full BTK occupancy of ibrutinib treated mice was validated using fluorescence resonance energy transfer-based assay. Ex vivo PDX MCL cells from PRT382-treated mice showed loss of symmetric dimethyl arginine with preservation of asymmetric dimethyl arginine levels, reduced H4(Sme2)R3 epigenetic marks, and elevated levels of BCL2, MYC, and pAKT/pERK. We developed a cell line (SEFA) allowing for in vitro mechanistic studies. We are currently investigating potential mechanisms responsible for circumventing IR-MCL by integrating genome-wide changes to chromatin accessibility and whole transcriptome analysis. This IR-MCL PDX mouse model serves as a useful tool to investigate mechanisms of drug resistance, provides a platform to explore novel pre-clinical therapeutic strategies to circumvent IR and demonstrates the therapeutic activity of PRMT5 targeted therapy in this aggressive disease. Disclosures Byrd: Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau. Vaddi:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

scholarly journals The Protein Kinase C Inhibitor MS-553 for the Treatment of Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2077-2077
Author(s):  
Elizabeth M. Muhowski ◽  
Amy M. Lehman ◽  
Sean D. Reiff ◽  
Janani Ravikrishnan ◽  
Rose Mantel ◽  
...  
Keyword(s):  
Research Funding ◽  
Ohio State University ◽  
Lymphocytic Leukemia ◽  
State University ◽  
Advisory Committees ◽  
University Patents ◽  
Kinase C ◽  
Hla Dr ◽  
Bcr Signaling ◽  
Equity Ownership

Introduction: Treatment of chronic lymphocytic leukemia (CLL) has been transformed by small molecule inhibitors targeting the B-cell receptor (BCR) signaling cascade. The first-in-class small molecule inhibitor of Bruton's Tyrosine Kinase (BTK), ibrutinib, is FDA approved as a frontline therapy for CLL. However, resistance to BTK inhibition has emerged in patients through acquisition of mutations in BTK or its immediate downstream target, PLCG2, emphasizing the need for alternative targets and therapies. BCR signaling remains intact in the presence of these mutations, making targeted inhibition of proteins downstream of BTK an attractive therapeutic strategy. Protein kinase C-β (PKCβ) is a downstream member of the BCR signaling pathway that we have previously demonstrated as an effective therapeutic target in CLL. MS-553 is a potent, ATP-competitive, reversible inhibitor of several PKC isoforms including PKCβ. Therefore, we evaluated the effects of MS-553 in primary CLL cells. Methods: Primary CLL cells were isolated by negative selection and treated with increasing concentrations of MS-553 to a maximum dose of 10 µM. BCR signaling changes were interrogated by change in target protein phosphorylation by immunoblot following a 24 hour drug incubation with and without phorbol ester stimulation (90 minutes) in CLL samples. Inhibition of CpG-mediated activation of CLL cells was measured using flow cytometry (CD86 and HLA-DR) in ibrutinib refractory patient samples at baseline and post-relapse due to the emergence of the p.C481S BTK mutation. CCL3 and CCL4 expression was measured by ELISA after 24 hours in primary CLL cells in the presence or absence of anti-IgM ligation. TNFα expression was also measured by ELISA in negatively selected, healthy donor T cells treated with MS-553 for 24 hours with or without anti-CD3 and anti-CD28 stimulation. Results: At 24 hours, 5 µM MS-553 inhibited downstream BCR signaling in primary CLL cells, demonstrated by 31% reduced phosphorylation of PKCβ (p=0.08, n=5) and several of its downstream targets including GSK3β (40%, p<.01, n=5) , ERK (46%, p=0.02, n=4) , and IκBα (56%, p=0.04, n=5) compared to vehicle treated, stimulated samples. CpG-mediated TLR9 stimulation increases expression of CD86 and HLA-DR in primary CLL cells. In baseline samples from ibrutinib treated patients, 10 µM MS-553 decreased expression of CD86 by 34% and HLA-DR by 91%. In matched patient samples post-relapse due to ibrutinib resistance, MS-553 (10 µM) maintained the ability to decrease expression of CD86 (49%) and HLA-DR (84%). Pro-inflammatory cytokine expression by primary CLL cells stimulated with anti-IgM decreased in the presence of 5 µM MS-553, with CCL3 decreasing by 36% (p=0.06, n=5) and CCL4 decreasing by 79% (p<.01, n=4) compared to vehicle treated, stimulated controls. TNFα expression by healthy T cells increased with anti-CD3 and anti-CD28 stimulation; 1 µM MS-553 reduced TNFα expression by 97% compared to vehicle treated, stimulated controls (p<.01, n=9). Conclusions: MS-553 is a novel and potent inhibitor of PKC demonstrating in vitro efficacy in CLL. MS-553 is able to inhibit BCR signaling by blocking phosphorylation of PKCβ and its downstream targets. CpG-mediated activation is reduced with MS-553 treatment in ibrutinib refractory patient samples both at baseline and post-relapse. Inflammatory signaling by primary CLL cells is further abrogated by MS-553 in its ability to decrease CCL3 and CCL4 cytokine expression. In an ongoing phase I clinical trial of MS-553, patient samples show a potent and dose dependent decrease in PKCβ activity as measured by a clinical biomarker assay. Together, our results suggest that MS-553 targets PKCβ in primary CLL to inhibit signaling and survival, establishing MS-553 as a potential therapeutic for treating CLL. These data justify continued preclinical and clinical work in the development of MS-553 for the treatment of CLL. Disclosures Niesman: MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zhang:MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Byrd:BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Genentech: Research Funding; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding. Woyach:Verastem: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding.

scholarly journals The Development of a Precision Medicine Platform to Overcome Ibrutinib Resistance in Mantle Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1487-1487
Author(s):  
Yang Liu ◽  
Shuangtao Zhao ◽  
Changying Jiang ◽  
Yixin Yao ◽  
Kelley Paige Murfin ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Akt Pathway ◽  
Therapeutic Resistance ◽  
Patient Sample ◽  
Mantle Cell ◽  
Btk Inhibitor ◽  
Ibrutinib Resistance

Background: While mantle cell lymphoma (MCL) initially responds to frontline therapies, this aggressive B-cell malignancy typically relapses or becomes resistant to treatment. Despite high overall response rates to the oral, covalent, first-in-class Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, most patients with relapsed/refractory MCL ultimately experience disease progression. To address the critical issue of BTK inhibitor resistance, novel therapies must be developed. Moreover, diversity in genetic alterations and the possibility that multiple pathway aberrations may contribute to disease progression and resistance makes overcoming this phenomenon with uniform treatment regimens extremely difficult, indicating that a personalized approach should be developed to overcome therapeutic resistance. In this study, molecular profiling of ibrutinib-resistant MCL patient samples has been conducted to identify targetable dysregulated signaling pathways and gene mutations associated with ibrutinib resistance. Clinical drug candidates targeting these potential pathways and their combinations with ibrutinib were analyzed in vitro to identify novel treatments that may potentially overcome ibrutinib resistance. Methods: Twenty-three agents targeting multiple pathways associated with ibrutinib resistance were deliberately chosen based on known targetable pathways in MCL to assess via high throughput drug screening. MCL tumor cells were isolated and purified from clinical apheresis and tumor excisional biopsies under established IRB-approved protocols. The same patient primary cells were subjected to gene expression analysis using a nanoString nCounter panel and whole exome sequencing (WES) to identify targetable dysregulated pathways and somatic mutations within each tumor. In vitro cell viability assays of single agents and drug combinations were tested per patient sample using the CellTiter-Glo luminescent assay (Promega), interrogating dysregulated pathways identified in each tumor. Subcutaneous, intravenous, and subrenal injections of the purified patient tumor cells were performed on NSG mice to create corresponding PDX mouse models for validation experiments. Results: nanoString nCounter analysis identified differentially expressed targetable pathways per patient sample such as BCR signaling, the PI3K/AKT pathway, NOTCH signaling, the cell cycle, and the NF-κB pathway. Correlations were identified between the WES and the nanoString nCounter analysis. For example, PTEN loss was observed in an MCL patient sample with high PI3K/AKT expression, demonstrating the potential underlying mechanism for the observed PI3K/AKT enrichment. Patients were divided into subgroups based on the identified responsive pathways in the in vitro screening. For example, PI3K/AKT pathway inhibitors were shown to be more potent against MCL samples in which the PI3K/AKT pathway was enriched. To further validate this finding, we created an MCL PDX model using a sample with enriched PI3K/AKT expression and successfully recapitulated the splenomegaly and hepatomegaly observed in the MCL patient. The MCL PDX mice were treated with the pan-PI3K inhibitor copanlisib (IP, 10 mg/kg) using a 5 on/2 off dosing schedule, which resulted in significantly reduced spleen (P &lt; 0.001) and liver size (P &lt; 0.01), as well as bone marrow involvement (P &lt; 0.05), compared with the vehicle control (Figure 1). Conclusions: Molecular matching with in vitro drug screening were utilized to develop a precision medicine platform for MCL to combat therapeutic resistance. This platform can be translated into a clinical setting to directly benefit the MCL patient population through treatment with therapies directly tailored to each patient. Disclosures Wang: Pulse Biosciences: Consultancy; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Acerta Pharma: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Dava Oncology: Honoraria; Kite Pharma: Consultancy, Research Funding; Juno Therapeutics: Research Funding; Celgene: Consultancy, Research Funding; MoreHealth: Consultancy, Equity Ownership; BioInvent: Consultancy, Research Funding; Aviara: Research Funding; BeiGene: Research Funding; Loxo Oncology: Research Funding; VelosBio: Research Funding.

scholarly journals LC-Facseq: A Novel Method for Detecting Rare Resistant Clones in Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3377-3377
Author(s):  
Eileen Hu ◽  
Hatice Gulcin Ozer ◽  
Arletta Lozanski ◽  
Tzyy-Jye Doong ◽  
Chi-Ling Chiang ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Cell Sorting ◽  
Research Funding ◽  
Clonal Evolution ◽  
Ohio State University ◽  
Lymphocytic Leukemia ◽  
State University ◽  
University Patents ◽  
Novel Method

Introduction: Targeted irreversible Bruton's Tyrosine Kinase (BTK) inhibitors ibrutinib and acalabrutinib, have revolutionized treatment for chronic lymphocytic leukemia (CLL). While BTK inhibition (BTKi) achieves durable responses in 90% of patients, only 10% achieve minimal residual disease (MRD) negative status. MRD positive patients have persistent residual CD5+CD19+ tumor B cells at approximately 1-5 /mm3 in peripheral blood. These cells may represent a subpopulation of B-cell lymphocytosis pre-malignant cells or may carry a BTK C481, PLCG2, or other CLL mutation that is ultimately responsible for disease relapse. Alternatively, MRD could be derived from the original clones present at initial disease presentation that are not dependent on BTK signaling. Readily available clinical DNA sequencing and MRD monitoring techniques lack the ability to characterize these cells adequately due to their rarity in peripheral blood. To address this problem, we developed a novel method for limited-cells using fluorescence activated cell sorting in tandem with next generation sequencing (LC-FACSeq) to characterize rare tumor subpopulations in the blood and bone marrow. LC-FACSeq may be useful not only for CLL but also other leukemias. Methods: LC-FACSeq uses fluorescent activated cell sorting (FACS) to isolate pure populations of rare tumor cells after which targeted deep sequencing is performed to monitor CLL-related mutations in NOTCH1, SF3B1, and TP53, as well as genes associated with BTKi relapse and resistance: BTK and PLCG2. For validation of this method, we generated libraries from DNA isolated from FACS isolated bulk (n >15000) versus n= 50, 100, 300, or 500 CD5+/CD19+ cells from CLL patients (n=5). Results: All samples analyzed had an average read depth of 1212 (SEM=56) per gene and an average coverage uniformity of 88.24% (SEM=.01). We show that showed that 300-cell LC-FACSeq libraries demonstrated comparable variant calling and minimal noise to standard libraries generated from purified DNA from bulk cells. Using samples from patients with previously identified BTK C481S mutations, we found that both sensitivity and specificity of LC-FACSeq for BTK C481S was 100%. Furthermore, LC-FACSeq reliably amplified BTK C481S signals from subclones as small as 6 in 300 total cells (2%) when mutated tumor cells were serially diluted into BTK wild type tumor cells. In using LC-FACSeq to retrospectively analyze four independent patients who developed Ibrutinib resistance, we found that we could see the emergence of small BTKi resistant subclones as early as 10 months before clinical detection. We next extended LC-FACSeq to examine the clonal architecture of long-term (> 12 months) ibrutinib-treated MRD positive patients. Median treatment time was 5 years. BTK C481S mutations were observed in the latest available on-treatment samples of only one patient. Using LC-FACSeq we observed canonical CLL-associated clonal mutations similar to those observed in previous studies. Of the 14 MRD positive patients, 7 showed subclonal changes in TP53, NOTCH1, POT1, SF3B1, and MYD88 over the course of ibrutinib treatment although we found no correlation or consensus in these clonal shifts. Conclusion: LC-FACSeq is a highly sensitive method of characterizing clonal evolution in rare cells. Our data shows that LC-FACSeq is useful for monitoring sequential acquisition of mutations conferring therapy resistance and clonal evolution in long-term ibrutinib treated chronic lymphocytic leukemia (CLL) patients. We also observe that in most cases, MRD clones after long-term ibrutinib treatment are genetically similar to disease clones from pretreatment baseline. Compared to current MRD monitoring strategies, the main advantages of LC-FACSeq are that 1) variants can be confidently called from rare sorted tumor populations and subpopulations, 2) library generation can be completed in less than a day in a diagnostic laboratory compared to the labor-intensive protocols of traditional NGS approaches, and 3) amplicon panels can be easily customized for application to other types of leukemia and lymphoma. (EH is supported by the Graduate Pelotonia Fellowship and the NIH F30) Disclosures Bhat: Janssen: Consultancy; Pharmacyclics: Consultancy. Rogers:Janssen: Research Funding; AbbVie: Research Funding; Genentech: Research Funding; Acerta Pharma: Consultancy. Woyach:AbbVie: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding. Lozanski:Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Genentec: Research Funding; Boehringer Ingelheim: Research Funding. Muthusamy:Ohio State University: Patents & Royalties: OSU-2S. Byrd:Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Genentech: Research Funding; Acerta: Research Funding.

Targeting Oxphos Pathway Against Ibrutinib Resistance to Mantle Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 290-290 ◽  
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.

scholarly journals Genomic Analysis of Cellular Hierarchy in Acute Myeloid Leukemia Using Ultrasensitive LC-FACS-Seq

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 186-186
Author(s):  
Caner Saygin ◽  
Arletta Lozanski ◽  
Tzyy-Jye Doong ◽  
Shelley Orwick ◽  
Deedra Nicolet ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Ohio State University ◽  
Median Frequency ◽  
State University ◽  
University Patents ◽  
Pre Treatment ◽  
Acute Myeloid

Normal hematopoiesis is organized in a hierarchical manner and it has been hypothesized that acute myeloid leukemia (AML) is organized in a similar way with leukemia-initiating cells (LIC) at the top of the hierarchy, giving rise to more differentiated blasts to sustain AML. Therefore, elimination of LIC population is critical for cure. This may be accomplished via novel molecular targeted therapies. The mutational composition of LIC and non-LIC compartments in AML has not been fully elucidated and could provide new insights into biology and treatment. We investigated the distribution and variant allelic frequencies (VAFs) of recurrent gene mutations within these compartments in newly diagnosed CD34+ AML patients (pts). We studied a total of 88 pts. CD34- AML cases, defined as &lt;5% positivity on blasts, were excluded. Pre-treatment bone marrow or apheresis samples were sorted and sequenced with our ultrasensitive limited cell (LC)-FACS-seq method. First, we gated on CD45dimLin- leukemic population, followed by isolation of 300 cells from CD34+CD38- (LIC), CD34+CD38+ (non-LIC) and CD34- compartments. To compare with the bulk population, DNA was extracted from 500,000 CD45dimLin- leukemic blasts. All samples were sequenced with a 27-gene targeted panel. Extreme Limited Dilution Analysis (ELDA) platform was used for colony formation assays and estimation of stem cell frequencies. Clinical characteristics are summarized in Table 1. The median frequency of the LIC population was 0.5% (range, 0.01% - 69%). The prevalence of high LIC frequency (≥0.5%) was significantly higher in pts with adverse risk (AR) AML, as compared to intermediate (IR) and favorable risk groups (94% vs 34% vs 16%, respectively, p&lt;.001). When compared to pts with low LIC frequency (&lt;0.5%), those with high LIC frequency had worse overall survival (median, 9 months vs not reached, p=.003) and relapse-free survival (median, 4 vs 15 months, p=.01). In 10 pts who had serial relapse samples, LIC frequencies were increased at the time of relapse (p=.03). We re-validated the commonly used LIC markers with ELDA of primary AML cells. In one IR sample, stem cell frequencies in sorted CD34+CD38-, CD34+CD38+ and CD34- compartments were 1:3, 1:15 and 1:16, respectively (p&lt;.001). In one AR sample, stem cell frequencies were 1:1, 1:8, and 1:12, respectively (p&lt;.001). Using these markers, LICs and non-LICs were enriched and sequenced. The average number of mutations detected by sequencing of bulk samples was significantly lower than sorted LIC (3.17 vs 3.75, p&lt;.05) and non-LIC (3.17 vs 3.96, p&lt;.001) populations indicating the higher sensitivity of our method in detecting subclonal mutations. Mean VAFs were similar between LIC and non-LIC populations for NPM1 (42% vs 47%), DNMT3A (37% vs 41%), IDH1 (41% vs 48%), IDH2 (43% vs 48%), and U2AF1 (37% vs 42%) mutations. Mutations involving signaling pathways were more frequent in non-LICs, including FLT3-TKD (12% vs 23%, p&lt;.01), NRAS (17% vs 26%, p&lt;.01) and KRAS (13% vs 19%, p&lt;.05) mutations, which might be explained by their later acquisition during AML development. In addition, among 22 pts with CEBPA mutation, 16 (73%) harbored the mutation exclusively in non-LICs. Finally, 13 pts with TP53 mutations had different VAFs between compartments. Among 4 pts who had doubling of VAF from LIC to non-LIC compartment, 3 had subclones with del(17p) in LIC pool detected by FISH. LIC subclones harboring both del(17p) and TP53 mutation (i.e. loss of heterozygosity) propagated to drive leukemia. Relapse samples obtained from 6 pts were analyzed and compared with diagnosis. In all cases, we could identify LIC clones that persisted after chemotherapy and led to relapse (see example in Figure). Similarly, 3 pts who were primary refractory showed persistence of LIC clones that were resistant to treatment. On the contrary, 6 pts in whom LIC clones could be eradicated with treatment did not experience disease recurrence. LICs exist at a very low frequency in pre-treatment AML samples. The mutational composition of LIC-enriched compartment shows differences from blasts constituting the bulk of leukemia, which is consistent with the sequence of mutations observed during the evolution of AML. LC-FACS-seq is an ultrasensitive method to detect mutations in a tiny population of residual LICs in pts at remission. Therapies targeting mutations that are concentrated in LICs may re-shape the clonal hierarchy and impact on disease course. Disclosures Behbehani: Fluidigm corporation: Other: Travel funding. Byrd:Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; Genentech: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Genentec: Research Funding.

scholarly journals Next-Generation RNA Sequencing-Based Analysis Identifies a Novel Set of Prognostic Micrornas (miRs) in Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2694-2694
Author(s):  
Dimitrios Papaioannou ◽  
Deedra Nicolet ◽  
Kellie J Archer ◽  
Allison Walker ◽  
Krzysztof Mrózek ◽  
...  
Keyword(s):  
Random Forest ◽  
Research Funding ◽  
Ohio State University ◽  
State University ◽  
Univariable Analysis ◽  
Younger Adults ◽  
Expression Levels ◽  
University Patents ◽  
Tcga Dataset ◽  
Cluster 2

Background: Aberrant expression levels of several miRs have been reported to independently associate with outcome of patients (pts) with CN-AML. In these reports, miR expression was profiled using microarray assays, which interrogate a selected subset of miRs. The advent of next-generation sequencing (NGS) has allowed unbiased measurement of miR expression, but, to our knowledge, NGS has not been used to identify miRs associated with prognosis of AML pts. Here, we analyze small RNA sequencing (smRNA-seq) data from a large cohort of younger adults with CN-AML, for whom outcome data were available, with the goal to identify new prognostic miRs. Methods: We performed smRNA-seq in 281 younger adults (aged 18-59 y) with de novo CN-AML. Cytogenetic analyses were performed in Cancer and Leukemia Group B (CALGB)/Alliance institutional laboratories using standard banding techniques; mutational analyses were done centrally using a targeted DNA sequencing platform. All pts were treated on frontline CALGB/Alliance protocols. Results: We first evaluated which miRs associated with overall survival (OS) in univariable analysis; we detected 9 such miRs. We then used a machine-learning approach, namely random forests, to identify miRs whose concomitant expression could generate an effective outcome predictor in CN-AML. To account for the effect of co-existing prognostic gene mutations, we included the European LeukemiaNet (ELN) risk group status in the random forest analyses. A total of 8 prognostic miRs were identified, 4 of which were also found to be prognostic in univariable analysis (underlined below), thus bringing the total number of unique prognostic miRs to 13: miR-511, miR-1193, miR-155, miR-4517, miR-3681, miR-2355, miR-628, miR-1266, miR-6715a, miR-1180, miR-6715b, miR-132, miR-146b. We used partitioning around medoids to divide our pts into clusters, based on the combined expression levels of the 13 prognostic miRs. Two such clusters were identified: cluster 1 comprised 173 pts and cluster 2 contained 108 pts. Regarding pretreatment and molecular features, pts in cluster 1 had lower percent of bone marrow blasts (P=.04), and had more frequently biallelic CEBPA mutations (P<.001) and less frequently internal tandem duplications of the FLT3 gene (P<.001), RUNX1 (P=.02) and WT1(P=.03) mutations than pts in cluster 2. In outcome analyses, pts in cluster 1 had a higher complete remission rate (CR; 91% vs 73%,P<.001) and a longer disease-free survival (DFS; 5-y rates 52% vs 16%, P<.001) and OS (5-y rates 60% vs 19%,P<.001). In multivariable analysis, cluster 1 status remained significantly associated with higher odds of achieving a CR (P=.001) and longer DFS (P<.001) and OS (P<.001), after adjusting for other covariates. Regarding accuracy of outcome prediction, our composite model had a concordance index of 0.687. When ranked according to importance for prognosis, miR-511 expression was the most significant determinant among the random forest model parameters. To evaluate the reproducibility of our findings, we performed analyses in the publicly available TCGA dataset (Ley et al. NEJM 2013;368:2059). Eighty-eight CN-AML pts with miR expression and survival data were available in the TCGA cohort. As TCGA pts are not classified according to ELN risk groups, we could not directly reproduce the random forest-based cluster analysis. However, a univariable analysis showed that miR-511 and miR-628 expression levels were also prognostic in the TCGA dataset. Next, we evaluated whether the identified prognostic miRs have functional relevance in AML. We focused on miR-511, which was the most important determinant of our outcome predictor and has not been previously studied in AML. Among 6 AML cell lines tested, MV4-11 had the most abundant expression of miR-511. Functional silencing of miR-511 in MV4-11 cells decreased both their viability (as measured by Annexin-PI staining and flow-cytometry, P=.004) and proliferative capacity (as measured by WST1 reagent degradation, P<.001). Conclusion: Unbiased profiling of miRs using smRNA-seq has identified a novel set of 13 miRs with prognostic significance in CN-AML. MiR expression-based cluster status independently associates with clinical outcome of CN-AML pts. Our preliminary in vitro experiments have shown that miR-511, whose association with prognosis was the strongest among the newly identified prognostic miRs, is functionally relevant in AML. Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Powell:Rafael Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding; Janssen: Research Funding. Kolitz:Astellas: Research Funding; Boeringer-Ingelheim: Research Funding; Roche: Research Funding. Byrd:Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau.

scholarly journals A Multicenter Study of Ibrutinib Resistance Development and Intervention with Venetoclax in Patients with Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3049-3049
Author(s):  
Kerry A Rogers ◽  
Lai Wei ◽  
Seema A. Bhat ◽  
Dan Jones ◽  
Erlene K. Seymour ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Research Funding ◽  
Response Assessment ◽  
Ohio State University ◽  
Lymphocytic Leukemia ◽  
State University ◽  
Phase 2 ◽  
University Patents ◽  
Secondary Endpoints

Background: Ibrutinib (ibr) for the treatment of chronic lymphocytic leukemia (CLL) has improved progression-free survival (PFS) compared to other treatments, especially in high-risk patients (pts). However, resistance occurs and is associated with mutations in the drug binding target (BTK) and its immediate downstream target (PLCg2). These ibr resistance mutations (IRmut) are detectable months prior to developing progressive disease (PD) and predict clinical relapse. Prospectively determining the time from starting ibr to development of IRmut and from IRmut detection to PD will improve our understanding of how to manage these patients. Venetoclax (ven) is highly effective after ibr and decreases IRmut. Adding ven to ibr for ibr resistance is a rational choice as this combination is safe and effective in CLL. Adding an agent rather than stopping ibr avoids disease flare associated with ibr discontinuation. This phase 2 study was designed to follow CLL pts taking ibr and at high risk for resistance (observation cohort) and to test ven in combination with ibr for those who develop PD (intervention cohort). This will determine: the incidence of IRmut and PD in this population, the ORR with ibr/ven, and the ability of this combination to eliminate IRmut. Trial Design and Methods: This multisite study will open at 4 centers initially. Eligible pts are adults with CLL taking ibr for ≥12 months and at high risk for ibr resistance defined as having ≥2 prior treatments and del(17p)(p13.1) on FISH panel and/or a complex karyotype. Pts with known IRmut or who cannot continue ibr for any reason are excluded. Enrolled pts enter the observation cohort and are followed every 3 months with a clinic visit, blood counts, and testing for IRmut. Pts who develop IRmut will also have CT scans at their visits to detect PD. Those with IRmut who develop PD by iwCLL 2018 criteria will enter the intervention cohort. Pts in the intervention cohort will start ven in addition to ibr. Ven will be ramped-up over 5 weeks to a target dose of 400mg. Pts will take combination ibr/ven for 12 cycles of 28 days in length. After 12 cycles they will undergo response assessment and those achieving a complete remission (CR) with no detectable leukemia (uMRD) in both the blood and bone marrow will stop ven and continue ibr alone. Those who do not achieve CR with uMRD will continue ibr/ven until cycle 24 and undergo a second response assessment. If in a CR with uMRD after 24 cycles they continue on ibr alone. If a CR with uMRD is not achieved after 24 cycles patients continue ibr/ven until PD, intolerance, death, or end of study which is 30 months after the last patient enters the intervention cohort (Figure). In the intervention cohort all pts will be tested for IRmut in the blood every 3 months with bone marrow testing at response assessments. The study has co-primary endpoints of ORR to combination ibr/ven after 12 cycles and the rate of IRmut negative status at that time in the intervention cohort. ORR will be tested first using a single-stage phase 2 design with a null hypothesis that the rate is ≤50% versus the alternative hypothesis that it is ≥75%. Only if the combination is effective in ORR will the rate of IRmut negative status be formally tested. Constraining overall Type I and II errors to 0.10 using this sequential testing strategy, 26 evaluable pts are required and 28 will be accrued. Secondary endpoints for the intervention cohort are the PFS and overall survival since starting the combination ibr/ven and the incidence and type of adverse events with ibr/ven. Secondary endpoints in the observation cohort are the incidence of IRmut during ibr treatment and the PFS after developing an IRmut. We estimate that 180 pt-years of follow up for the observation cohort will be needed. The yearly rate of mutation development in this population is approximately 20%, therefore this will identify 36 pts with IRmut. Of those with IRmut, approximately 80% will remain eligible to enter the intervention cohort. Accrual to the observation cohort will stop once 28 pts enter the intervention cohort. Conclusion: This multicenter phase 2 trial examines the development of IRmut and clinical resistance to ibr in a cohort of high-risk CLL pts and will determine the efficacy of adding ven to ibr in those who develop PD. We expect to determine the natural course of molecular and clinical ibr resistance in CLL and if adding ven is an effective treatment strategy. Figure Disclosures Rogers: Acerta: Consultancy; Genentech: Research Funding; Abbvie: Research Funding; Janssen: Research Funding. Bhat:Pharmacyclics: Consultancy; Janssen: Consultancy. Stephens:Karyopharm: Research Funding; Gilead: Research Funding; Acerta: Research Funding. Ye:Janssen: Research Funding; Karyopharm: Research Funding; Portola: Research Funding; MingSight: Research Funding; Sanofi: Research Funding. Byrd:Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Genentech: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding. Woyach:Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding. OffLabel Disclosure: This abstract discussion the use of combination ibrutinib and venetoclax in CLL.

scholarly journals ROR1 Targeted Immunoliposomal Delivery of OSU-2S Show Selective Cytotoxicity in t(1;19) Translocated B-ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3798-3798
Author(s):  
Swagata Goswami ◽  
Chi-Ling Chiang ◽  
Kevan Zapolnik ◽  
Zhiliang Xie ◽  
James L. Lee ◽  
...  
Keyword(s):  
Research Funding ◽  
Hematological Malignancies ◽  
Cell Lymphoma ◽  
Tumor Burden ◽  
Leukemic Cells ◽  
State University ◽  
Research Support ◽  
University Patents ◽  
Relative Viability

The receptor tyrosine kinase ROR1 is uniquely expressed on and required for many hematological malignancies such as t(1;19) positive acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). The t(1;19) is one of the most frequent translocations in B-ALL, observed in both adult and pediatric patients. The translocation has intermediate prognosis on its own, but is associated with a poor prognosis in the unbalanced der(19)t(1;19) form in pediatric ALL, and in the context of hyperdiploid B-ALL. While leukemic cell dependence on ROR1 is known, ROR1 lacks kinase activity making it difficult to target therapeutically. However, we have previously shown that ROR1 can be targeted to deliver therapeutic payload specifically to leukemic cells in CLL, sparing the normal cells from toxic side effects. This encouraged us to develop ROR1 directed immunoliposomal nanoparticles encapsulating a novel small molecule OSU-2S. OSU-2S is a non-immunosuppressive derivative of the sphingosine analogue FTY720, with potent anti-tumor activity against multiple hematological malignancies including CLL, mantle cell lymphoma (MCL) and canine B-cell lymphoma. OSU-2S demonstrated potent dose dependent cytotoxicity in patient derived B-ALL samples with different cytogenetic backgrounds including translocations t(4;11), t(9;22) and t(1;19) as well as hyperdiploid, hypodiploid and normal cytogenetic background [n=7, p= 0.0032 (0 vs 2.5µM), mean decrease in relative viability= 44.51±12.12%] as assessed by Annexin V/Propidium Iodide staining. We confirmed ROR1 expression on t(1;19) translocated patient samples by flow cytometry, and synthesized ROR1 targeted OSU-2S immunoliposomal nanoparticles (2A2-OSU-2S-ILP) (mean size= 186.9 +/- 0.8 nm, mean concentration= 1.38*1013 particles/ml). 2A2-OSU-2S-ILP was selectively cytotoxic to t(1;19) translocated ALL, including unbalanced der(19)t(1;19), from relapsed patients aged 29-37, but not ROR1-ve, t(1;19) non translocated ALL, as compared to control IgG-OSU-2S-ILP, or 2A2/IgG immunoliposomes without OSU-2S [n=3, p= 0.04, mean decrease in relative viability (IgG-OSU-2S-ILP vs 2A2-OSU-2S-ILP)= 35.14±7.36%]. Similar results were seen in ROR1+ve 697 cells, a B-ALL cell line carrying t(1;19) translocation, where 2A2-OSU-2S-ILP showed selective cytotoxicity [n=8, p=0.004, mean decrease in relative viability (IgG-OSU-2S-ILP vs 2A2-OSU-2S-ILP)= 61.62±14.63%]. To assess the effect of 2A2-OSU-2S-ILP on t(1;19) positive ALL in-vivo, we used a disseminated cell line derived xenograft model. Immunocompromised NSG mice were engrafted with 697 cells, treated with 2A2-OSU-2S-ILP or IgG-OSU-2S-ILP for 14 days and tumor burden was assessed in the spleen and bone marrow. 2A2-OSU-2S-ILP treatment significantly reduced the number of human CD45+CD19+ cells in the bone marrow as compared to IgG-OSU-2S-ILP cohort (n=6 per cohort, p=0.022, mean decrease in 697 cells in marrow= 1.751 ± 0.6372 million cells/ femur). There was also a trend towards decreased tumor burden in spleen (mean decrease in 697 cells in spleen= 1.883 ± 0.9729 million cells). Together, these data show the ability of ROR1 targeted liposomal nanoparticles to selectively deliver its payload to leukemic cells in t(1;19) translocated B-ALL, sparing toxicity to the normal cells. Ongoing studies are directed towards understanding the mechanistic basis of OSU-2S mediated therapeutic benefit in B-ALL in-vitro and in-vivo. [This work was supported by NIH-R01-CA197844-01. SG is supported by Pelotonia Graduate Fellowship] Disclosures Baskar: NIH: Patents & Royalties: ROR1 mAb 2A2. Rader:NIH: Patents & Royalties: ROR1 mAb 2A2. Byrd:Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau. Bhatnagar:Novartis and Astellas: Consultancy, Honoraria; Cell Therapeutics, Inc.: Other: Research support; Karyopharm Therapeutics: Other: Research support. Muthusamy:Ohio State University: Patents & Royalties: OSU-2S.

Genetic and Molecular Characterization of Ibrutinib-Resistant Mantle Cell Lymphoma: Designing Innovative Therapeutic Strategies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1838-1838 ◽  
Author(s):  
Hui Guo ◽  
Shengjian Huang ◽  
Yang Liu ◽  
Carrie J Li ◽  
Jack Wang ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Therapeutic Strategies ◽  
Rna Seq ◽  
Biological Functions ◽  
Advisory Committees ◽  
Mantle Cell ◽  
Resistant Patient ◽  
Ibrutinib Resistance

Abstract Background Mantle cell lymphoma (MCL) is a B-cell malignancy with a broad spectrum of clinical, pathological, and biological features, and clinical evolution is usually very aggressive with short responses to treatment and frequent relapses. Ibrutinib is considered the drug of choice in relapsed-refractory cases but patients may develop resistance as some patients in complete remission (CR) have not relapsed yet and are in long term follow-up. The understanding of the resistance mechanisms and the emergence of new drugs targeting key oncogenic mechanisms are providing the basis for designing innovative therapeutic strategies to overcome ibrutinib resistance, both in preclinical studies and preliminary clinical trials. Methods We identified differentially expressed genes (DEGs) in 7 ibrutinib-primary resistant MCL patient samples compared with 16 ibrutinib-sensitive MCL patient samples by next generation sequencing (RNA-Seq) and top28-gene signature were developed via Gene Set Enrichment Analysis (GSEA) analysis of RNA-Seq data, and we also verified the expression level of these genes in ibrutinib-resistant and-sensitive MCL patient samples using Real time-PCR, and then a secondary focus of this study was to identify potential predictive biomarkers for therapy in relapsed or refractory MCL. In order to place the gene expression data into a biological context, Ingenuity Pathway Analysis (IPA) software was used to assign the DEGs to know the canonical pathways and functional networks in order to predict the biological functions of the transcriptional changes. Results We identified top-28 DEGs in five ibrutinib-resistant MCL patient samples compared with four ibrutinib-sensitive MCL patient samples. We performed gene enrichment and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG pathway) of differentially expressed genes of each samples and verified the predicted genes using Real time-PCR, which were truly related to MCL and not false positive results. Moreover, Using IPA software, we identified that the enriched biological functions in the MCL ibrutinib-resistant patient samples were Oxidative phosphorylation, Mitochondrial Dysfunction and TCA Cycle (Eukaryotic), which were enriched biological functions in the analysis of RNA-Seq data, and which may be targeted by oncogenic events in MCL, and they may influence the tumor response to new therapeutic agents. We also found that expression of these genes (SEPT3, FASN, IDH3A, SLC1A5, INPP5J, CCT5, MTHFD1) was significantly increased in five ibrutinib-resistant MCL patient samples compared with four ibrutinib-sensitive MCL patient samples, and we used IPA to identify some functionally related genes with these genes increased in MCL ibrutinib-resistant patient samples and built networks based on the molecular relationships most relevant to this project. Conclusion These data identify a genomic basis for ibrutinib-primary resistance in MCL and provide the important insights into the strategy to address the problem of ibrutinib-resistance, and will hopefully allow more tailored and specific therapies to be designed. Disclosures Wang: Onyx: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Kite Pharma: Research Funding; Asana BioSciences: Research Funding; BeiGene: Research Funding; Juno Therapeutics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Oncogenic MALT1 Promotes Cell Survival and Mediates Ibrutinib Resistance and Ibrutinib-Venetoclax Resistance in Mantle Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 18-18
Author(s):  
Vivian Changying Jiang ◽  
Junwei Lian ◽  
Shengjian Huang ◽  
Shaojun Zhang ◽  
Yang Liu ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Mtor Signaling ◽  
Mantle Cell ◽  
Whole Transcriptome Sequencing ◽  
Ibrutinib Resistance ◽  
Whole Transcriptome

Both as monotherapies and in combination, the Bruton's tyrosine kinase inhibitor ibrutinib and the BH3 mimetic BCL2 inhibitor venetoclax have proven to be efficacious and are now widely used treatment options for mantle cell lymphoma (MCL) patients. However, mono- and dual- resistance frequently develops, necessitating investigation into the mechanisms mediating resistance to these therapies. To investigate the mechanism of ibrutinib resistance, we generated two ibrutinib-resistant cells due to marked BTK knockdown via CRISPR/CAS9 from JeKo-1, which is ibrutinib-sensitive and venetoclax-resistant. To understand the mechanism of venetoclax resistance, we generated three venetoclax-resistant cell lines with acquired resistance via chronic exposure to increasing doses of venetoclax from ibrutinib/venetoclax double sensitive Mino and Rec-1 cells, and ibrutinib-resistant but venetoclax-sensitive Granta519 cells. All these paired cell lines with various resistance to ibrutinib and venetoclax were subject to whole transcriptome sequencing of these paired MCL cell lines. We discovered that mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is significantly overexpressed in ibrutinib-resistant and ibrutinib-venetoclax dual-resistant MCL cells, especially in cells with BTK knockdown. This was further validated in primary MCL patient cells (n=24). Interestingly, MALT1 overexpression inversely correlates with CARD11 expression and enhances non-canonical NF-κB signaling, suggesting a switch from a highly-dependent BTK-CARD11 mechanism to an independent mechanism in both ibrutinib-resistant and ibrutinib-venetoclax dual-resistant MCL cells. Chromosomal translocations of MALT1 are the hallmarks of MALT lymphoma, which result in oncogenic fusion of MALT1 products. MALT1 is constitutively active in driving aggressive ABC-type DLBCL. This indicates MALT1 can be oncogenic when its activity is dysregulated. Therefore, we hypothesized that constitutive MALT1 activity may be responsible for the resistance to ibrutinib and venetoclax in MCL cells. To demonstrate this relationship, MALT1 ablation using genetic manipulation resulted in significant growth defects both in vitro and in vivo. Pharmaceutical approaches using MALT1 inhibitor MI-2 resulted in similar effects on cell survival and using cell viability assays. Whole transcriptome sequencing analysis revealed that MYC, NF-kB, ROS, cell cycle and mTOR signaling are the most significantly downregulated pathways upon MI-2 treatment. Intriguingly, MYC, NF-kB, PI3K-AKT-mTOR and mTOR signaling pathways were reported to be upregulated in ibrutinib-resistant MCL cells compared to sensitive MCL cells. To address this further, proteomics analysis by reverse phase protein array (RPPA) using more than 400 antibodies confirmed that MI-2 significantly downregulated AKT-mTOR signaling. NF-kB modulation, ROS production, AKT-mTOR, and metabolism changes were further confirmed through multiple biochemical approaches. In addition, MI-2 treatment resulted in a dramatic reduction of MALT1 expression, suggesting that MI-2 treatment affected both its scaffold and paracaspase activities. Furthermore, MI-2 treatment resulted in significant inhibition of in vivo tumor growth of ibrutinib-venetoclax dual-resistant MCL subcutaneous xenografts and tumor homing to the spleen and bone marrow in an ibrutinib-venetoclax dual-resistant MCL patient-derived xenograft (PDX) mouse model. In conclusion, we discovered that MALT1, an essential regulator of NF-κB signaling, is hyperactive in ibrutinib-resistant cells and ibrutinib-venetoclax dual-resistant MCL cells, which puts MALT1 forward as a potentially new therapeutic target in ibrutinib and venetoclax-resistant MCL tumors. Genetic depletion or pharmaceutical inhibition of MALT1 resulted in remarkable defects in cell survival and cell proliferation. The MALT1 inhibitor MI-2 proved its in vivo potency by its pro-apoptotic effect and its significant tumor growth inhibition. In conclusion, targeting a hyperactive MALT1 is a promising therapeutic strategy that could lead to clinical implementation of a new treatment strategy meant to overcome ibrutinib and ibrutinib-venetoclax dual resistance in MCL patients by reversing the NF-kB and ROS/mTOR- mediated resistance in these tumors. Disclosures Wang: Targeted Oncology: Honoraria; Loxo Oncology: Consultancy, Research Funding; 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; InnoCare: Consultancy; Oncternal: Consultancy, Research Funding; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; MoreHealth: Consultancy; 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; OMI: Honoraria, Other: Travel, accommodation, expenses.

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