A Novel Evolutionary Pattern Revealed Using Deep Sequencing of Immunoglobulin Loci at Diagnosis and over the Course of Treatment in Multiple Myeloma Patients

Abstract Introduction: Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. As multiple myeloma is considered a clonal disease originating from the transformation of a single plasma cell, myeloma cells are traditionally thought to have one clonal Ig gene sequence that remains stable throughout the course of the disease. We previously observed that multiple Ig sequences related by somatic hypermutation (SHM) may be present in some MM patients at diagnosis. Here we provide an expanded observation in a very large cohort of the patients, and perform mutational analysis of the oligoclonal myeloma clonotypes observed at diagnosis and post-treatment, revealing changes in the relative frequency of the MM clonotypes and emergence of new Ig clones. Methods : 620 MM patientsenrolled in IFM/DFCI and Hospital 12 de Octubre trials were included in this analysis. The next-generation sequencing (NGS)-based immunosequencing platform was used to detect evidence of oligoclonality at the Ig heavy chain loci. Using universal primer sets, we amplified IGH variable, diversity, and joining gene segments from DNA and/or RNA isolated from purified CD138+ MM cells collected at the time of diagnosis. MM-specific clonotypes were identified for each patient based on their high frequency (5%) within the B-cell repertoire in the diagnostic (dx) sample. The highest frequency MM clonotype in a dx sample is termed the "index clonotype." DNA and/or RNA isolated from dx AND post-treatment bone marrow samples were assessed for evidence of evolved MM clonotypes. Results: We identified Ig clones in 367 RNA samples and 430 DNA samples from the cohort. We first looked for cases with evidence that myeloma cells have two unrelated origins. We found 11/620 (1.8%) cases at diagnosis, which had evidence of unrelated clones as evident by having three IgH or two functional sequences. In 8 of the 11 cases (72.6%), we had multiple samples to analyze, including two samples at diagnosis or diagnosis/post-treatment pairs. In 4 of the 8 samples, we saw dramatically different relative frequencies of unrelated clones in these samples suggesting that these unrelated clones are likely to be present in two distinct cells. We then considered cases where we found two IgH sequences that are related to each other by SHM at diagnosis. Overall 79 (12.7%) of 620 samples had more than one evolved clone; of these 63/367 (17.2%) of RNA dx samples showed evidence of evolved clones via SHM, while 22/430 patients (5.1%) showed evidence of evolved clones related to the index clone via SHM in DNA samples from diagnosis. Mutant clonotypes had an average of 3.9 to 4.5 mutations in the CDR3 region. We also noted mixed isotypes in 13 clones from 13 patients at diagnosis. The majority of related clones observed in the RNA samples are present at very low frequencies (<10-4), as the greater sequencing depth in RNA allows for identification of low frequency clones. 304 post-treatment samples from 206 patients were MRD positive and were assessed for the presence of clonal evolution. In 27/304 follow-up samples (8.8%) and 7/206 patients (3.4%), an evolved clone related to the index clone was observed even though the period between diagnosis and post-treatment samples was only 6 months. In 6 patients, a substantial change in the relative index and unrelated clone frequencies was observed from the dx to post-treatment time points suggesting a differential sensitivity to treatment. Conclusions: We confirm presence of multiple evolved clonotypes in a substantial percentage of diagnostic MM samples in a large cohort of patients. The evolution of multiple clones related by SHM indicates that SHM remains active after myeloma development and may also impact other non-Ig sites. These findings shed light on the biology and pathogenesis of MM and may provide prognostic information. The very high depth of our sequencing also indicates that the emergence of new IgH clones may be newly acquired mutations in the Ig gene, driven by some ongoing genomic mutation process. Thus, these evolved myeloma clonotypes may be useful as surrogate markers for other oncogenic mutations providing resistance to therapy. Disclosures Munshi: Takeda: Consultancy; Pfizer: Consultancy; Merck: Consultancy; Celgene Corporation: Consultancy; Oncopep: Consultancy, Equity Ownership. Carlton:Adaptive Biotechnologies: Employment, Equity Ownership. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Attal:amgen: Consultancy, Research Funding; celgene: Consultancy, Research Funding; janssen: Consultancy, Research Funding; sanofi: Consultancy. Moreau:Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria, Speakers Bureau; Novartis: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sonofi Aventis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Other: Scientific Founder; Oncopep: Other: Scientific Founder. Faham:Adaptive Biotechnologies Corp: Employment. Avet-Loiseau:janssen: Consultancy; sanofi: Consultancy; celgene: Consultancy; amgen: Consultancy.

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Deep Igh Sequencing Identifies an Ongoing Somatic Hypermutation Process with Complex and Evolving Clonal Architecture in Myeloma

Blood ◽

10.1182/blood.v126.23.21.21 ◽

2015 ◽

Vol 126 (23) ◽

pp. 21-21 ◽

Cited By ~ 7

Author(s):

Nikhil C. Munshi ◽

Stephane Minvielle ◽

Yu-Tzu Tai ◽

Mariateresa Fulciniti ◽

Mehmet K Samur ◽

...

Keyword(s):

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Somatic Hypermutation ◽

Post Treatment ◽

Differential Sensitivity ◽

Advisory Committees ◽

Maintenance Time ◽

Equity Ownership ◽

Time Point

Abstract Introduction: Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. Multiple myeloma (MM), a malignancy of plasma cells, typically have mutated Ig sequences but are thought to be stable throughout the course of the disease. We previously observed that multiple Ig sequences related by somatic hypermutation (SHM) may be present in some MM patients at diagnosis. Here we provide an expanded observation and investigate whether there is ongoing evolution in Ig sequences over the course of the disease. Methods: 550 MM patientsenrolled in IFM/DFCI study were included in this analysis. The next-generation sequencing (NGS)-based immunosequencing platform was used to detect evidence of oligoclonality at the Ig heavy chain loci. Using universal primer sets, we amplified IGH variable, diversity, and joining gene segments from DNA and/or RNA isolated from purified CD138+ MM cells collected at the time of diagnosis. Amplified products were sequenced and analyzed (Faham et al., Blood 2012). MM-specific clonotypes were identified for each patient based on their high frequency (5%) within the B-cell repertoire in the diagnostic (dx) sample. The highest frequency MM clonotype in a dx sample is termed the Òindex clonotype.Ó DNA and/or RNA isolated from dx AND post-treatment bone marrow samples were assessed for evidence of evolved MM clonotypes. A clonotype was considered ÒevolvedÓ based on CDR3 sequence homology to the dx Òindex clonotype.Ó Results: We identified Ig clones in 340 RNA samples and 311 DNA samples from the IFM/DFCI cohort.We first looked at V segment usage in these MM clones comparedto a database of ~30 million Ig VDJ sequences derived from normal B cells. The frequencies for 6 V segments were found to be significantly different from this dataset compared to the database.We then looked for cases with evidence that Myeloma cells have two unrelated origins. We found 9/550 (1.6%) cases which had evidence of unrelated clones as evident by having three IgH or two functional sequences. We then considered cases where we find two IgH sequences that are related to each other by SHM. 128/340 (37.6%) of RNA dx samples showed evidence of evolved clones via SHM, with 69/128 (53.9%) having 3 or more related clones, while 15/311 patients (4.8%) showed evidence of evolved clones related to the index clone via SHM in DNA samples from diagnosis. Of note, the majority of RNA evolved clones were found at low frequency (<10-3) which would have been impossible to observe in the limited cell input DNA samples available for testing. Out of the 15 patients with evidence of evolved clones related to the index clone, we tested RNA from 8 of them. In 4/8 cases, the index and the related clones were present at a similar ratio in the DNA and RNA, while in 3/8 cases the index clone was found in the RNA but not the related clone. 249 post-treatment samples from 164 patients were MRD positive and were assessed for the presence of clonal evolution. In 19/249 follow-up samples (7.6%), an evolved clone related to the index clone was observed. In 6/19 patients, a substantial change in the relative index and evolved clone frequencies was observed from the dx to post-treatment time points suggesting a differential sensitivity to treatment. For example, in one case, the evolved ÒnewÓ clonotype was not observed at diagnosis but appears in the post-maintenance sample only. In another case, the evolved clonotype either increased or decreased in the post-maintenance sample relative to the index clonotype (Fig 1). Conclusions: We observed multiple evolved clonotypes in a substantial percentage of dx MM samples (37.6%). The presence of multiple clonotypes related by SHM indicates that this mechanism remains active after myeloma development in at least a portion of the cells. We also found marked changes in the relative frequency of the MM clonotypes in post-treatment samples and emergence of new Ig clones which may not be due to selective advantage of the newly acquired mutations in the Ig gene, but rather some other ongoing genomic mutation process. Thus, these evolved myeloma clonotypes may be useful as surrogate markers for other oncogenic mutations providing resistance to therapy. Figure 1. Evolution of related clones in the post-maintenance time point. Below are the sequences of two related clones, with one base difference bolded and underlined. Figure 1. Evolution of related clones in the post-maintenance time point. Below are the sequences of two related clones, with one base difference bolded and underlined. Disclosures Richardson: Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees. Attal:celgene: Membership on an entity's Board of Directors or advisory committees; jansen: Honoraria. Anderson:Celgene Corporation: Consultancy; Millennium: Consultancy; BMS: Consultancy; Gilead: Consultancy; acetylon pharmaceuticals: Equity Ownership; Oncocorp: Equity Ownership. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder. Avet-Loiseau:BMS: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

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Targeting Brouton's Tyrosine Kinase with PCI-32765 Blocks Growth and Survival of Multiple Myeloma and Waldenström Macroglobulinemia Via Potent Inhibition of Osteoclastogenesis, Cytokines/Chemokine Secretion, and Myeloma Stem-Like Cells in the Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v118.21.883.883 ◽

2011 ◽

Vol 118 (21) ◽

pp. 883-883

Author(s):

Yu-Tzu Tai ◽

Betty Y Chang ◽

Sun-Young Kong ◽

Mariateresa Fulciniti ◽

Guang Yang ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tyrosine Kinase ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Growth And Survival ◽

Equity Ownership ◽

Trap Staining

Abstract Abstract 883 Specific expression of Bruton's tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in regulating osteoclastogenesis. Although Btk is critical in B cell maturation and myeloid function, it has not been characterized in plasma cell malignancies including multiple myeloma (MM) and Waldenström Macroglobulinemia (WM). We here investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) microenvironment, as well as on MM and WM cancer cells. We further define molecular targets of Btk signaling cascade in OCs and MM in the BM milieu. In CD14+ OC precursor cells, RANKL and M-CSF stimulate phosphorylation of Btk in a time-dependent fashion; conversely, PCI-32765 abrogates RANKL/M-CSF-induced activation of Btk and downstream PLCγ2. Importantly, PCI-32765 decreased number of multinucleated OC (>3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and the secretion of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It increased size of OCs and number of nuclei per OC, with significantly defective bone resorption activity as evidenced by diminished pit formation on dentine slices. Moreover, lack of effect of Dexamethasone on OC activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 significantly reduced cytokine and chemokine secretion from OC cultures, including MIP1α, MIP1β, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1–0.48 nM). It potently decreased IL-6, SDF-1, MIP1α, MIP1β, and M-CSF in CD138-negative cell cultures from active MM patients, associated with decreased TRAP staining in a dose-dependent manner. In MM and WM cells, immunoblotting analysis confirmed a higher Btk expression in CD138+ cells from majority of MM patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated a higher expression of Btk and its downstream signaling components in WM cells than in CD19+ normal bone marrow cells. PCI-32765 significantly inhibits SDF-1-induced adhesion and migration of MM cells. It further blocked cytokine expression (MIP1a, MIP-1β) at mRNA level in MM and WM tumor cells, correlated with inhibition of Btk-mediated pPLCγ2, pERK and NF-kB activation. Importantly, PCI-32765 inhibited growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs in IL-6-dependent INA6 and ANBL6 MM cells. Furthermore, myeloma stem-like cells express Btk and PCI-32765 (10–100 nM) blocks their abilities to form colonies from MM patients (n=5). In contrast, PCI-32765 has no adverse effects on Btk-negative BMSCs and OBs, as well as Btk-expressing dendritic cells. Finally, oral administration of PCI-32765 (12 mg/kg) in mice significantly suppresses MM cell growth (p< 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results provide compelling evidence to target Btk in the BM microenvironment against MM and WM., strongly supporting clinical trials of PCI-32765 to improve patient outcome in MM and WM. Disclosures: Chang: Pharmacyclics Inc: Employment. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership. Elias:Pharmacyclics Inc: Consultancy. Treon:Millennium: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Identification and Validation of HLA-A24 XBP1us, XBP1sp, CD138, and CS1 Peptides to Generate Antigens Specific-Cytotoxic T Lymphocytes: Preclinical Basis for Vaccine Therapy in HLA-A24 Patients with Multiple Myeloma and Other Cancers

Blood ◽

10.1182/blood.v128.22.5689.5689 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5689-5689

Author(s):

Jooeun Bae ◽

Brandon Nguyen ◽

Matthew Ho ◽

Yan Song ◽

Noopur Raje ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Tumor Cells ◽

Cytotoxic T Lymphocytes ◽

Research Funding ◽

Checkpoint Inhibitors ◽

Preclinical Studies ◽

Advisory Committees ◽

Myeloma Cells ◽

Equity Ownership

Abstract XBP1 (X-box binding protein 1), CD138 (Syndecan-1), and CS1 (CD2 subset 1, CRACC, SLAMF7, CD319) are highly expressed antigens associated with tumor pathogenesis in multiple myeloma cells and various solid tumor cells. We have previously reported on the use of HLA-A2 peptides specific to these antigens to generate antigen-specific and HLA-2-restricted cytotoxic T cells (CTL) in preclinical studies and phase I/II clinical trials in patients with smoldering MM. These studies extend this concept to HLA-A24 patients. Immunogenic HLA-A24 peptides specific to both the spliced and unspliced isoforms of XBP1, CD138, and CS1 were identified, synthesized, and evaluated in vitro for their ability to generate the antigen-specific cytotoxic T lymphocytes (CTL) targeting tumor cells. The specific epitopes were selected based on their binding affinity to HLA-A24, extended half-time disassociation rates, proteasomal C terminal cleavage, and TAP transport. We report here on novel highly immunogenic HLA-A24-specific peptides XBP1 US185 -193 (I S P W I L A V L), XBP1 SP223-231 (V Y P E G P S S L), CD138265-273 (I F A V C L V G F), and CS1240-248 (L F V L G L F L W), which induce antigen-specific CD3+CD8+ CTL against MM and various solid tumors. Each of the single peptide-specific CTL displayed immune functional activities including IFN-g/IL-2 cytokine production and cytotoxic activity against HLA-A24+ MM cells. Furthermore, our studies demonstrated that a cocktail of the four HLA-A24-specific peptides induced multipeptide-specific CD3+CD8+ CTL with functional anti-tumor properties against myeloma cells. Phenotypically, the multipeptide-specific CTL were central memory (CM; CCR7+CD45RO+/CD3+CD8+), effector memory (EM; CCR7-CD45RO+/CD3+CD8+) and terminal effector (TE; CCR7-CD45RO-/CD3+CD8+) cells expressing high levels of cell activation markers CD69, CD38, and CD40L. The multipeptide-specific effector and memory CTL subsets demonstrated HLA-A24-restricted and peptide-specific anti-tumor activities, and the highest activities were detected within the CM CTL subset. Treatment of the multipeptide-specific CTL or tumor target cells with different types of checkpoint inhibitors (a-PD1, a-TIM3, a-PDL-1, a-Galectin-9) or the immune modulator drug, lenalidomide, enhanced their anti-myeloma activities and antigen-specific CD3+CD8+ T cell proliferation to HLA-A24+ tumor cells. Therefore, a cocktail of the four immunogenic HLA-A24 epitopes provides the framework for development of a cancer vaccine based targeted immunotherapy. In conclusion, we report here on novel immunogenic HLA-A24-specific XBP1 unspliced, XBP1 spliced, CD138, and CS1 peptides that elicit CTL with anti-myeloma activities. These peptides will allow us to extend our immunotherapeutic vaccine approach beyond the current HLA-A2-specific peptides to HLA-A24 patients with smoldering myeloma, multiple myeloma, other plasma cell disorders and potentially with solid tumors. These preclinical studies also demonstrate that incorporation of immunomodulatory agents and/or checkpoint inhibitors can amplify and maintain antigen-specific memory CTL responses against tumor. Disclosures Bae: OncoPep Inc.: Consultancy, Equity Ownership. Raje:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Eli Lilly: Research Funding. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Hideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Chauhan:Stemline Therapeutics: Consultancy. Munshi:OncoPep Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Anderson:Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

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Results of a Phase I Study of Pnk-007, Allogeneic, Off the Shelf NK Cell, Post Autologous Transplant in Multiple Myeloma (NCT02955550)

Blood ◽

10.1182/blood-2019-128467 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4451-4451

Author(s):

Sarah A. Holstein ◽

Sarah Cooley ◽

Parameswaran Hari ◽

Sundar Jagannath ◽

Catherine R Balint ◽

...

Keyword(s):

Multiple Myeloma ◽

Maintenance Therapy ◽

Board Of Directors ◽

Research Funding ◽

Phase I Study ◽

Nk Cell ◽

Single Infusion ◽

Advisory Committees ◽

Cd34 Cells ◽

Equity Ownership

Background: PNK-007 is an allogeneic, off the shelf cell therapy product enriched for CD56+/CD3- NK cells expanded from placental CD34+ cells. PNK-007 cells exhibit cytotoxicity against various cancer cell types, including multiple myeloma (MM), and secrete cytokines during co-culture with cancer cells. This is a Phase I study of single infusion PNK-007 after autologous stem cell transplant (ASCT) in MM. Methods: Placental CD34+ cells were cultivated in the presence of cytokines for 35 days to generate PNK-007 under cGMP standards followed by release testing. HLA matching and KIR mismatching were not used. Four treatment arms were evaluated on patients (pts) following ASCT: 10 million (M) cells/kg Day (D) 14 with or without recombinant human IL-2 (rhIL-2), 30M cells/kg D14 with rhIL-2, or 30M cells/kg D7 with rhIL-2. rhIL-2 was administered subcutaneously at 6M units every other day for up to 6 doses to facilitate PNK-007 expansion. Pts received variable pre-ASCT induction therapy. Maintenance therapy was permitted after the Day 90-100 visit (D90). Subjects were followed for up to 1-year. Results: 15 pts who received PNK-007 (12 of whom received rhIL-2) were followed on this study. Pts aged 44-69 yrs included 12 newly diagnosed (ND)MM and 3 relapsed/refractory (RR)MM. The 3 RRMM pts had received 1, 2 or 5 prior lines of therapy, with 2 pts having previous ASCT. All pts had been exposed to immunomodulatory drug (IMiDs) and proteasome inhibitors (PIs). No serious adverse events (AEs) were attributable to PNK-007 and no dose-limiting toxicity, GvHD, graft failure or graft rejection were observed. 12/15 pts started maintenance therapy following the transplant while participating in this study, at the physician's discretion. Based on physician assessed responses by International Myeloma Working Group pre-ASCT, of the NDMM pts 10/12 achieved VGPR or better (1 CR and 9 VGPR), 1/12 achieved PR and 1/12 was not assessed during pre-ASCT induction. By D90 10/12 pts achieved VGPR or better (5 CR or sCR and 5 VGPR), 1/12 maintained PR and 1/12 stable disease. At 1-year 9/11 achieved VGPR or better (4 CR or sCR and 5 VGPR), 2/11 were not assessed and 1 was removed from the study prior to 1 year due to failure to respond to ASCT. Of the RRMM pts 2/3 achieved PR and 1/3 was not assessed during pre-ASCT induction, by D90 2/3 achieved VGPR and the pt that had not been assessed pre-ASCT achieved PR. At 1-year, 1 pt maintained VGPR, 1 pt was not assessed and 1 pt did not continue to the 1-year visit. Using a validated Euro-flow minimal residual disease (MRD) assay of bone marrow aspirate (BMA) samples, of the NDMM pts 4/12 were MRD negative (MRD-) pre-ASCT; by D90 9/12 were MRD-. At 1-year 6/12 were MRD-, 2/12 had insufficient BMA to perform testing, 2/12 refused BMA procedure, 1/12 did not convert to MRD-, and 1 was removed from the study prior to 1-year due to failure to respond to ASCT. Of the RRMM pts 0/3 were MRD- pre-ASCT with 1/3 having insufficient BMA to perform testing; by D90 1/3 were MRD-. At 1-year 1/3 was MRD-, 1/3 did not convert to MRD- and 1 pt did not continue to the 1-year visit. PNK-007 infusion did not interfere with immune reconstitution kinetics. Platelet, neutrophil, and absolute lymphocyte counts recovered by day 28 post-ASCT in 12/15 patients. All pts' sera tested negative for the presence of anti-HLA antibodies at all timepoints indicating the absence of humoral immunity and alloantibodies to PNK-007. Conclusion: PNK-007 is the first fully allogeneic, off the shelf CD34+ derived NK cell product in MM clinical trials. A single infusion of PNK-007 up to 30M cells/kg with and without rhIL-2 was well tolerated in the post-ASCT setting. We established the feasibility of infusing PNK-007 as early as 7 days post-ASCT without negative impact on blood count recovery or successful engraftment. BMA MRD- status was observed in 7/9 MRD evaluable pts at 1-year post ASCT. These clinical data are encouraging and warrant further evaluation. Disclosures Holstein: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Sorrento: Consultancy; GSK: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees. Cooley:Fate Therapeutics, Inc: Employment, Equity Ownership. Hari:Cell Vault: Equity Ownership; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Kite: Consultancy, Honoraria; Amgen: Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria. Jagannath:BMS: Consultancy; Merck: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Medicom: Speakers Bureau; Multiple Myeloma Research Foundation: Speakers Bureau. Balint:Celgene: Equity Ownership; Celularity, Inc: Employment. Van Der Touw:Celularity, Inc: Employment. Zhang:Celularity Inc: Employment. Hariri:Celularity Inc: Employment. Vij:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Honoraria; Karyopharm: Honoraria; Sanofi: Honoraria; Takeda: Honoraria, Research Funding.

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Phase II Trial of the Combination of Ixazomib, Lenalidomide, and Dexamethasone in High-Risk Smoldering Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117871 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 804-804 ◽

Cited By ~ 7

Author(s):

Mark Bustoros ◽

Chia-jen Liu ◽

Kaitlen Reyes ◽

Kalvis Hornburg ◽

Kathleen Guimond ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

High Risk ◽

Disease Progression ◽

Board Of Directors ◽

Rna Sequencing ◽

Research Funding ◽

Response Rate ◽

Advisory Committees ◽

Equity Ownership

Abstract Background. This study aimed to determine the progression-free survival and response rate using early therapeutic intervention in patients with high-risk smoldering multiple myeloma (SMM) using the combination of ixazomib, lenalidomide, and dexamethasone. Methods. Patients enrolled on study met eligibility for high-risk SMM based on the newly defined criteria proposed by Rajkumar et al., Blood 2014. The treatment plan was designed to be administered on an outpatient basis where patients receive 9 cycles of induction therapy of ixazomib (4mg) at days 1, 8, and 15, in combination with lenalidomide (25mg) at days 1-21 and Dexamethasone at days 1, 8, 15, and 22. This induction phase is followed by ixazomib (4mg) and lenalidomide (15mg) maintenance for another 15 cycles. A treatment cycle is defined as 28 consecutive days, and therapy is administered for a total of 24 cycles total. Bone marrow samples from all patients were obtained before starting therapy for baseline assessment, whole exome sequencing (WES), and RNA sequencing of plasma and bone marrow microenvironment cells. Moreover, blood samples were obtained at screening and before each cycle to isolate cell-free DNA (cfDNA) and circulating tumor cells (CTCs). Stem cell collection is planned for all eligible patients. Results. In total, 26 of the planned 56 patients were enrolled in this study from February 2017 to April 2018. The median age of the patients enrolled was 63 years (range, 41 to 73) with 12 males (46.2%). Interphase fluorescence in situ hybridization (iFISH) was successful in 18 patients. High-risk cytogenetics (defined as the presence of t(4;14), 17p deletion, and 1q gain) were found in 11 patients (61.1%). The median number of cycles completed was 8 cycles (3-15). The most common toxicities were fatigue (69.6%), followed by rash (56.5%), and neutropenia (56.5%). The most common grade 3 adverse events were hypophosphatemia (13%), leukopenia (13%), and neutropenia (8.7%). One patient had grade 4 neutropenia during treatment. Additionally, grade 4 hyperglycemia occurred in another patient. As of this abstract date, the overall response rate (partial response or better) in participants who had at least 3 cycles of treatment was 89% (23/26), with 5 Complete Responses (CR, 19.2%), 9 very good partial responses (VGPR, 34.6%), 9 partial responses (34.6%), and 3 Minimal Responses (MR, 11.5%). None of the patients have shown progression to overt MM to date. Correlative studies including WES of plasma cells and single-cell RNA sequencing of the bone microenvironment cells are ongoing to identify the genomic and transcriptomic predictors for the differential response to therapy as well as for disease evolution. Furthermore, we are analyzing the cfDNA and CTCs of the patients at different time points to investigate their use in monitoring minimal residual disease and disease progression. Conclusion. The combination of ixazomib, lenalidomide, and dexamethasone is an effective and well-tolerated intervention in high-risk smoldering myeloma. The high response rate, convenient schedule with minimal toxicity observed to date are promising in this patient population at high risk of progression to symptomatic disease. Further studies and longer follow up for disease progression are warranted. Disclosures Bustoros: Dava Oncology: Honoraria. Munshi:OncoPep: Other: Board of director. Anderson:C4 Therapeutics: Equity Ownership; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Takeda Millennium: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; BMS: Consultancy.

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Chromosomal Translocation t(11;14) Induced By the Cre-Loxp System in Normal B Cell-Derived Ips Cells for the Study of Myeloma-Initiating Cells

Blood ◽

10.1182/blood-2020-134572 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 18-19

Author(s):

Misaki Sugai ◽

Naohiro Tsuyama ◽

Yu Abe ◽

Yusuke Azami ◽

Kenichi Kudo ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Board Of Directors ◽

B Lymphocytes ◽

Research Funding ◽

Chromosomal Translocation ◽

Stem Cell Differentiation ◽

Advisory Committees ◽

Myeloma Cells ◽

Company Limited

The cellular origin of multiple myeloma (MM) has not yet been identified. Based on immunoglobulin heavy chain (IgH) gene analysis, myeloma cells are derived from mature B cells. Chromosomal aberrations such as trisomy and chromosomal translocation (cTr) play a critical role in the early tumorigenesis of MM. We hypothesized that the abnormal cells from which myeloma cells originate might be mature B lymphocytes with chromosomal or genetic changes in the reprogrammed state that enable them to acquire the potential to become tumors in the process of redifferentiation into B lymphocytes. We established induced pluripotent stem cells (iPSs) from normal B lymphocytes (BiPSCs: BiPSC13 & MIB2-6); these BiPSCs have the same VDJ rearrangement of IgH as the original B lymphocytes and differentiate into CD34+/CD38- hematopoietic progenitor cells co-culture with stromal cells, AGM-S3 (Sci Rep, 2017). We then established a method to induce reciprocal cTr t(11;14), which is a reciprocal cTr between IgH and CCND1 and the most frequent cTr in MM, using the CRISPR/Cas9 system; cTr was induced by infection of IgH-CCND1 lentiCRISPRv2 lentivirus, which targets the human IgH Eµ region and 13kb upstream of the CCND1 coding sequence, to BiPSCs (Oncol Lett, 2019). Subsequently, we established cell lines carrying reciprocal cTr t(11;14) between CCND1 and either an allele in which VDJ rearrangement of IgH had been completed or an allele in which VDJ rearrangement had not been completed (stopped at DJ joining) in BiPSC13 t(11;14) (AZ & AX) and MIB2-6 t(11;14) (BC & BG), respectively. These BiPSCs differentiated into CD34+/CD38-/CD45+/-/CD43+/- hematopoietic progenitors cells in co-culture with AGM-S3 or in stem cell differentiation medium; this was subsequently confirmed by the differentiation into granulocytes, macrophages, and erythroblasts in a colony-formation assay. We are now trying to produce BiPSCs in which cTr t(11;14) is induced when they differentiate into mature B cells expressing CD27. First, we used the Cre-loxP recombination system to induce cTr t(11;14) in BiPSCs. BiPSCs were transfected with IgH loxP-Neo-loxP knock-in vector and IgH lentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying loxP-Neo-loxP in IgH were transfected with iCre-EGFP. After removing the loxP-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP were transfected with CCND1 loxP-FRT3-Neo-FRT3 knock-in vector and CCND1 lentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying IgH-loxP in IgH and loxP-FRT3-Neo-FRT3 in CCND1 were transfected with Flpo-EGFP. After removing the FRT3-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP in IgH and CCND1-loxP-FRT3 in CCND1 were transfected with iCre-HygR. Hygromycin B-resistant cells were picked, the reciprocal cTr t(11;14) was confirmed by polymerase chain reaction, and we established BiPSCs with der(11)t(11;14) and BiPSCs with der(14)t(11;14). We also developed a system in which Cre is expressed along with CD27 expression in the B cell lymphoma cell line Raji. These BiPSCs could be useful for the study of myeloma-initiating cells, but whether they would be able to be redifferentiated into B lymphocyte is important. Disclosures Hanamura: Mundipharma K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SHIONOGI Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis Pharma K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; DAIICHI SANKYO COMPANY, LIMITED: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eisai Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NIPPON SHINYAKU CO.,LTD.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer Japan Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda Pharmaceutical Company Limited: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Ono Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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A Phase I Study of Bendamustine Combined with Lenalidomide and Dexamethasone in Patients with Refractory or Relapsed Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.1856.1856 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1856-1856 ◽

Cited By ~ 1

Author(s):

Suzanne Lentzsch ◽

Amy O’Sullivan ◽

Silvana Lalo ◽

Carrie Kruppa ◽

Diane Gardner ◽

...

Keyword(s):

Multiple Myeloma ◽

Partial Response ◽

Board Of Directors ◽

Dose Level ◽

Research Funding ◽

Advisory Board ◽

Clinical Activity ◽

Advisory Committees ◽

Equity Ownership ◽

Grade 3

Abstract Abstract 1856 Poster Board I-882 Background: Lenalidomide is an analog of thalidomide that has shown significant clinical activity in patients with relapsed or refractory multiple myeloma (MM), both as a single agent and in combination with dexamethasone. Bendamustine is a bifunctional alkylating agent that is approved for the treatment of chronic lymphocytic leukemia and indolent non-Hodgkin's lymphoma that has progressed during or relapsed within 6 months following a rituximab-containing regimen. Bendamustine combined with lenalidomide may be an effective treatment option for MM patients, particularly those with preexisting or bortezomib-induced neuropathy. Our primary objective was to determine the maximum tolerated dose (MTD) and safety profile of bendamustine and lenalidomide when administered with dexamethasone for patients with relapsed or refractory MM. Methods: Patients aged ≥18 years with confirmed, measurable stage 2 or 3 MM that was refractory to or progressed after 1 or more prior therapies, including lenalidomide, received bendamustine by intravenous infusion on days 1 and 2, oral lenalidomide on days 1–21, and oral dexamethasone on days 1, 8, 15, and 22 of each 28-day cycle. Treatment was continued until a plateau of best response, as determined by the IBMTR/ABMTR, was reached. Study drug doses were escalated through 4 levels (Table), with 3–6 patients enrolled at each level depending on the rate of dose-limiting toxicity (DLT). After determining the MTD, up to an additional 12 patients will be enrolled in an MTD expansion arm to better evaluate toxicity and clinical activity. Secondary endpoints included preliminary efficacy, as evidenced by objective response, time to disease progression, and overall survival. Results: To date, 11 patients have been enrolled, with a median age of 63 years (range, 38–75 years). The MTD of bendamustine and lenalidomide has not been identified at this point; currently, patients are enrolling on dose level 3 with 100 mg/m2 bendamustine and 10 mg lenalidomide. Thus far, DLT included 1 grade 4 neutropenia at dose level 2. Nine of 11 patients are currently eligible for response assessment. A partial response was observed in 67% of patients, including 1 very good partial response and 5 partial responses (PR). Two patients experienced stable disease and 1 exhibited progressive disease. Grade 3/4 adverse events included grade 3 neutropenia, thrombocytopenia, anemia, hyperglycemia, and prolonged QTC, and 1 grade 4 neutropenia. Conclusions: Bendamustine, lenalidomide, and dexamethasone form a well-tolerated and highly active regimen even in heavily pretreated MM patients, with a PR rate of 67%. Additional updates on response and MTD will be available at the time of presentation. Disclosures: Lentzsch: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cephalon: Consultancy, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Bendamustine is not FDA approved for the treatment of multiple myeloma in the USA. Burt:Millennium: Honoraria; Celgene: Honoraria. Mapara:Resolvyx: Consultancy, Research Funding; Genzyme: Membership on an entity's Board of Directors or advisory committees; Gentium: Equity Ownership; Celgene: Spouse is consultant , has received research funding, and participates on advisory board; Cephalon: Spouse has received funding for clinical trial and participates on advisory board. Redner:Biogen: Equity Ownership; Wyeth: Equity Ownership; Glaxo-Smith-Kline: Equity Ownership; Pfizer: Equity Ownership; Genzyme: Membership on an entity's Board of Directors or advisory committees. Roodman:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy; Acceleron: Consultancy. Zonder:Amgen: Consultancy; Pfizer: Consultancy; Cephalon: Consultancy; Millennium: Consultancy, Speaking (CME only); no promotional talks.

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A Phase I Study of Vorinostat, Lenalidomide, and Dexamethasone In Patients with Relapsed or Relapsed and Refractory Multiple Myeloma: Excellent Tolerability and Promising Activity In a Heavily Pretreated Population

Blood ◽

10.1182/blood.v116.21.1951.1951 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1951-1951 ◽

Cited By ~ 4

Author(s):

Paul Richardson ◽

Donna Weber ◽

Constantine S. Mitsiades ◽

Meletios A. Dimopoulos ◽

Jean-Luc Harousseau ◽

...

Keyword(s):

Multiple Myeloma ◽

Phase I ◽

Board Of Directors ◽

Research Funding ◽

Phase I Study ◽

Response Rate ◽

Advisory Board ◽

Advisory Committees ◽

Equity Ownership ◽

Grade 3

Abstract Abstract 1951 Background: Although novel treatment combinations for multiple myeloma (MM) have improved outcomes, the disease remains incurable and new drug combinations are urgently needed. Vorinostat is an oral histone deacetylase inhibitor approved in the United States for treatment of patients (pts) with advanced cutaneous T-cell lymphoma who failed prior therapies. Vorinostat alters gene expression and protein activity, promoting MM cell death through multiple pathways, and has been shown in preclinical studies to synergistically enhance the anti-MM activity of bortezomib and immunomodulatory drugs, including lenalidomide, with or without dexamethasone. Aims: The primary objective of this Phase I study was to determine the maximum tolerated dose (MTD) of vorinostat plus lenalidomide and dexamethasone in pts with relapsed or relapsed and refractory MM. Secondary objectives included overall safety, tolerability, response rate, duration of response, and time to progression (TTP). Methods: Pts in this Phase I multicenter open-label study were sequentially enrolled into 1 of 5 escalating doses of the combination regimen using a standard 3 + 3 design for ≤8 cycles. Pts who tolerated treatment and experienced clinical benefit were eligible for enrollment in an extension phase. Toxicity was evaluated using the National Cancer Institute Common Terminology Criteria (version 3.0). Response was assessed using the modified European Group for Blood and Marrow Transplantation criteria and International Myeloma Working Group Uniform Criteria. Safety and efficacy data were analyzed using summary statistics, except for TTP, which was estimated by the Kaplan-Meier method. Results: As of July 15, 2010, 31 pts were treated and evaluable for toxicity; 4 pts remain on study. Most pts had received prior thalidomide (n=22; 71%), bortezomib (n=20; 65%), or lenalidomide (n=14; 45%), with a median of 4 prior therapies (range, 1–10). The patient population contained both high-risk and low-risk pts, based on cytogenetic and/or fluorescence in situ hybridization analyses. Most adverse events (AEs) were mild or moderate in severity. The most common grade ≥3 treatment-related AEs, experienced by 19 (61%) pts, were neutropenia (26%), thrombocytopenia (16%), diarrhea (13%), anemia (10%), and fatigue (10%); 8 pts discontinued due to toxicity. One dose-limiting toxicity (grade 3 diarrhea lasting >48 h) was observed at the maximum assessed dose (level 5), but MTD was not reached (Table) and there were no treatment-related deaths. Among 30 pts evaluable for response, the median TTP was 32 weeks (5 mo), and 4 pts remain on study as of the data cutoff date; 26 of 30 pts (87%) have achieved at least stable disease (SD). Best single responses included 2 complete responses, 3 very good partial responses (VGPR), 11 partial responses (PR), and 5 minimal responses (MR), with 5 pts achieving SD and 4 developing progressive disease, resulting in an overall response rate (ORR; PR or better) of 53%. Of 13 evaluable pts who had previously received lenalidomide, a best single response of SD or better was observed in 9 (69%; 2 VGPR, 3 PR, 1 MR, 3 SD), resulting in a 38% ORR. Notably, SD or better (2 PR, 1 MR, 3 SD) was observed in 60% of 10 evaluable pts who were relapsed, refractory, or intolerant to previous lenalidomide-containing regimens. Conclusions: Preliminary data from this Phase I study suggest that vorinostat plus lenalidomide and dexamethasone is a convenient and generally well-tolerated regimen with promising activity for relapsed or relapsed and refractory MM. The MTD for this combination was not reached. Importantly, responses were observed in pts who had received prior lenalidomide, bortezomib, and thalidomide. Further evaluation of this regimen is planned in future trials. Disclosures: Richardson: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Off Label Use: Vorinostat, Lenalidomide, and Dexamethasone for treatment in Multiple Myeloma. Weber:Novartis-unpaid consultant: Consultancy; Merck- unpaid consultant: Consultancy; Celgene- none for at least 2 years: Honoraria; Millenium-none for 2 years: Honoraria; Celgene, Millenium, Merck: Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck & Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding. Dimopoulos:MSD: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Harousseau:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Houp:Merck Research Laboratories: Employment. Graef:Merck Research Laboratories: Employment. Gause:Merck Research Laboratories: Employment. Byrne:Celgene Corporation: Employment, Equity Ownership. Anderson:Millennium Pharmaceuticals: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; BMS: Consultancy; Acetylon: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Siegel:Celgene and Millennium: Advisory Board, Speakers Bureau; Merck: Advisory Board.

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Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells

Blood ◽

10.1182/blood.v118.21.136.136 ◽

2011 ◽

Vol 118 (21) ◽

pp. 136-136

Author(s):

Ze Tian ◽

Jian-Jun Zhao ◽

Jianhong Lin ◽

Dharminder Chauhan ◽

Kenneth C. Anderson

Keyword(s):

Multiple Myeloma ◽

Tumor Suppressor ◽

Board Of Directors ◽

Expression Profiling ◽

Research Funding ◽

Advisory Committees ◽

Myeloma Cells ◽

Investigational Agent ◽

Reporter Activity

Abstract Abstract 136 Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells Ze Tian, Jianjun Zhao, Jianhong Lin, Dharminder Chauhan, Kenneth C. Anderson Medical Oncology, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA, 02115 MicroRNAs (miRNAs) are 19–25 nucleotide-long noncoding RNA molecules that regulate gene expression both at the level of messenger RNA degradation and translation. Emerging evidence shows that miRNAs play a critical role in tumor pathogenesis by functioning as either oncogene or tumor suppressor genes. The role of miRNA and their regulation in response to proteasome inhibitors treatment in Multiple Myeloma (MM) is unclear. Here, we utilized MLN9708, a selective orally bio-available proteasome inhibitor to examine its effects on miRNA alterations in MM.1S MM cells. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to its biologically active form MLN2238. Our previous study using both in vitro and in vivo models showed that MLN2238 inhibits tumor growth and triggers apoptosis via activation of caspases. Moreover, MLN2238 triggered apoptosis in bortezomib-resistant MM cells, and induced synergistic anti-MM activity when combined with HDAC inhibitor SAHA, dexamethasone, and lenalidomide. In the current study, we treated MM.1S cells with MLN2238 (12 nM) for 3 hours and harvested; total RNA was subjected to miRNA profiling using TaqMan® Array Human miRNA A-Card Set v3.0 and the data was analyzed using dChip analysis. Results showed that MLN2238 modulates miRNA expression with a total of 36 miRNA changing their expression profiling (δδCT>1.5 or δδCT <-1.5; 19 were upregulated and 17 showed a downregulation). Among all miRNA, miR-33b was highly (δδCT>7) upregulated in response to MLN2238 treatment. We therefore hypothesized that miR-33b may play a role in MM pathogenesis as well as during MLN2238-induced proteasome inhibition in MM cells. We first utilized quantitative polymerase chain reaction (q-PCR) to validate the changes in miRNA expression profiling. Results confirmed that MLN2238 treatment triggers significant increase in the miR-33b expression in MM.1S cells (2.1 and 2.2 folds at 3h and 6h, respectively; P<0.001). Examination of normal PBMCs and plasma cells showed higher expression of miR-33b than patient MM cells (P<0.001). We further investigated the functional role of miR-33b in MM cells at baseline and during MLN2238 treatment. Drug sensitivity, cell viability, apoptosis, colony formation, and migration assays were performed using cell TilTer-Glo, Annexin V-FITC/PI staining, MTT staining, and Transwell assays, respectively. Signaling pathways modulated post miR-33b overexpression were evaluated by q-PCR, immunoblot, and reporter assays. Our findings show that overexpression of miR-33b significantly decreased cell viability, cell migration, colony formation, as well as increased apoptosis and sensitivity of MM cells to MLN2238 treatment. Targetscan analysis predicted pim-1 as a putative downstream target of miR-33b. Overexpression of miR-33b downregulated pim-1 mRNA and protein expression. To further corroborate these data, we co-tranfected miR-33b and Pim-1-wt or Pim-1-mt in 293T and MM.1S cell lines. In concert with our earlier findings, miR-33b decreases pim-1-wt, but not pim-1-mt reporter activity in both cell lines. Reflecting the overexpression study results, MLN2238 treatment also decreases pim-1-wt, but not pim1-mt reporter activity. Moreover, a biochemical inhibitor of pim1/2 triggered apoptosis in MM cells. Finally, overexpression of miR-33b inhibits tumor growth (P<0.001) and prolongs survival (P<0.001) in both subcutaneous and disseminated human MM xenograft models. In summary, our study suggests that miR-33b is a tumor suppressor, which plays a role during MLN2238-induced apoptotic signaling in MM cells, and provide the basis for novel therapeutic strategies targeting miR-33b in MM. Disclosures: Anderson: Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership.

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DEPTOR Is a Regulator of Response to mTOR Kinase Inhibitors in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.2916.2916 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2916-2916

Author(s):

Diana Cirstea ◽

Teru Hideshima ◽

Loredana Santo ◽

Homare Eda ◽

Miriam Canavese ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Research Funding ◽

Kinase Inhibitors ◽

Mtor Inhibitors ◽

Mtor Pathway ◽

Differential Sensitivity ◽

Advisory Committees ◽

Mtor Kinase ◽

Regulatory Cascade

Abstract Abstract 2916 Inhibition of the PI3K/mTOR pathway is a promising therapeutic strategy in targeting multiple myeloma (MM) cells in the bone marrow (BM) microenvironment, which abnormally activates PI3K/mTOR signaling cascade mediating proliferation, anti-apoptosis and drug resistance. Exploring the targeting of PI3K/mTOR pathway has led to the development of different therapeutic approaches; however, mTORC1 inhibitors (i.e., temsirolimus and everolimus) have demonstrated only modest activity as single agents. In this regard, several mechanisms underlying rapamycin resistance, including mTOR/S6K1-mediated feedback loops resulting in activation of PI3K/Akt and ERK signaling, have been proposed. Importantly, recent studies have identified mTOR kinase and the mTOR-DEPTOR counter-regulatory cascade as key mediators of mTORC1 and mTORC2 multi-protein complexes, with differential sensitivity to rapamycin. Indeed, targeting DEPTOR/mTORC1/mTORC2 signaling by inhibition of mTOR kinase proved an effective strategy to overcome some of the limitations of TORC1 inhibition in MM cells, evidenced in our studies of the novel dual mTORC1 and mTORC2 selective inhibitor AZD8055. Unlike rapamycin, AZD8055 induced apoptosis and inhibited MM cell growth even when co-cultured with cytokines (i.e., IL-6, IGF1) or BMSCs, presumably through simultaneous suppression of mTORC1 and mTORC2 signaling including the rapamycin-resistant 4E-BP1 (downstream of mTORC1) and Akt as well as NDRG1 (effectors of mTORC2). We examined mRNA and protein level of DEPTOR in MM cell lines treated with AZD8055 versus rapamycin and observed no significant changes. To examine the functional significance of DEPTOR in response to mTOR inhibitors, we utilized lentiviral shRNA to knockdown DEPTOR in OPM1 MM cells. DEPTOR-knockdown cells acquired resistance to AZD8055 treatment, suggesting that DEPTOR is a key modulator of mTORC1/2 signaling. Moreover, DEPTOR knockdown triggered decrease in Akt phosphorylation (Ser473), associated with suppression of Rictor phosphorylation (Thr1135). DEPTOR co-immunoprecipitation with Rictor was also abrogated by both AZD8055 and rapamycin treatment. Taken together, our results indicate the role of DEPTOR, either alone or as an mTOR/Rictor interacting molecule, in mediating the anti-MM activity induced by mTOR kinase inhibitors in MM cells. These data therefore both provide insights into the molecular profiles that may predict sensitivity/resistance to second generation of mTOR inhibitors in MM, and may be useful to select MM patients for mTOR inhibitor therapy. Disclosures: Hideshima: Acetylon: Consultancy. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees. Guichard:AstraZeneca, UK: Employment, Shares from AstraZeneca, UK. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

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