Mutations in CRBN and Other Cereblon Pathway Genes Are Only Associated with Acquired Resistance to Immunomodulatory Drugs in a Subset of Patients and Cell Line Models

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-7
Author(s):  
Amy Barber ◽  
John R Jones ◽  
Harvey Che ◽  
Yann-Vai Le Bihan ◽  
Niels Weinhold ◽  
...  
Keyword(s):  
Cell Line ◽  
Board Of Directors ◽  
Research Funding ◽  
Cross Resistance ◽  
Immunomodulatory Drugs ◽  
Private Company ◽  
Advisory Committees ◽  
Data Set ◽  
Synonymous Mutations

Background: Immunomodulatory drugs (IMiDs) are the current backbone of standard and experimental combination myeloma therapies at all stages of disease, but the majority of patients eventually relapse. The mechanisms driving IMiD resistance are poorly understood. Previous studies looking for genetic drivers of resistance have looked at core members of the CRL4CRBN E3-ubiquitin ligase complex (CUL4-RBX1-DDB1-CRBN) and identified infrequent mutations and deletions in cereblon (CRBN), but at a rate that cannot account for resistance in the majority of patients. More recently several in vitro studies have identified novel regulators of cereblon activity including the COP9 signalosome, E2 ubiquitin conjugating enzymes, neddylation modifiers and additional IMiD neosubstrates. In this study paired presentation/relapse samples from newly diagnosed patients recruited to a clinical trial (UK NCRI Myeloma XI trial: NCT01554852) of largely IMiD-based therapies were used to investigate the role of mutations and deletions in all genes implicated in IMiD activity. For comparison, cell line models of resistance were generated in vitro. Methods: 56 patients who received IMiD induction therapy followed by either lenalidomide maintenance (n=30) or observation (n=26), and subsequently relapsed, underwent whole exome sequencing (WES) of CD138+ cells, median depth 122x for tumour samples and 58x for paired germline controls. Non-synonymous mutations and deletions present in tumour but not germline controls were considered. Cell line models were generated using the IMiD sensitive MM1s cell line. Cells were cultured in 10xGI50 concentrations of lenalidomide/pomalidomide alongside a control exposed to the same %DMSO. WES was carried out and non-synonymous mutations identified. Mutations present in the lenalidomide resistant (Len-R) and pomalidomide resistant (Pom-R) but not their relevant DMSO exposed control were considered. From recent publications a list of 42 genes (Figure 1) involved in cereblon pathway regulation and IMiD response was curated, termed "CRBN/IMiD genes". Mutations in CRBN/IMiD genes in the patient dataset and cell line models were examined. Results: In the patient data set 12/42 (28.6%) of the CRBN/IMiD genes were found to be mutated, with a total of 17 mutations in 14/56 (25%) patients identified. 9/17 (53%) were identified in patients who had received lenalidomide maintenance and 8/17 (47%) in the observation group. Importantly, in the patients receiving lenalidomide maintenance, 6 of the 9 (66.7%) mutations had a higher cancer clonal fraction (CCF) at relapse, suggesting they may have been selected for by exposure to treatment. Comparatively, in mutations identified in patients undergoing observation, only 3 of the 8 (37.5%) mutations had a higher CCF at relapse compared with presentation. The only deletion in CRBN/IMiD genes was in SETX, in one patient at relapse. Only one mutation or deletion was identified in CRBN itself, a missense mutation at relapse at g.3:3195148A>C, encoding a Cys326Gly sequence modification at the protein level. Interestingly, Cys326 is one of 4 cysteines in CRBN coordinating a single zinc ion to form a Zn finger motif, which stabilises the Thalidomide Binding Domain (TBD) of the protein, suggesting this mutation may have had functional significance. In the cell line models full resistance up to 100xGI50 concentrations was established by 12 weeks. The resistant cell lines had cross-resistance to the other IMiDs and comparable morphology, growth rates and responses to non-IMiD drugs as their sensitive counterpart. Resistant cells had reduced levels of CRBN mRNA and protein expression. Functional assays demonstrated that well characterised downstream effects of IMiD treatment were abrogated: transcription factors Ikaros and Aiolos not degraded and no downregulation of IRF4 mRNA. The Pom-R cell line had a mutation affecting a CRBN splice site 5' of exon 8. No other mutations or deletions in the 42 IMiD pathway genes were identified in either the Len-R or Pom-R lines. Conclusions: CRBN and other genes in the IMiD response pathway were mutated or deleted in around 25% of patients suggesting other mechanisms, for example epigenetic alterations, underlie resistance acquisition in a significant proportion. Models for both CRBN/IMiD gene mutated and unmutated resistant states have been generated and will be used to study mechanisms of IMiD resistance. Figure Disclosures Jones: Celgene: Honoraria, Research Funding. Che:Monte Rosa Therapeutics: Research Funding. Le Bihan:Monte Rosa Therapeutics: Research Funding. Wang:Monte Rosa Therapeutics: Research Funding. Kaiser:Bristol-Myers Squibb, Chugai, Janssen, Amgen, Takeda, Celgene, AbbVie, Karyopharm, GlaxoSmithKline: Consultancy; Janssen, Amgen, Celgene, Bristol-Myers Squibb, Takeda: Honoraria; Bristol-Myers Squibb/Celgene, Janssen, Karyopharm: Research Funding; Bristol-Myers Squibb, Takeda: Other: Travel expenses. Jackson:Takeda: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Gsk: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau. Davies:Celgene/BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotech: Honoraria; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Chopra:Apple Tree Life Sciences: Current Employment; Monte Rosa Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Research Funding. Morgan:GSK: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Pawlyn:Takeda: Consultancy, Other: Travel expenses; Celgene: Consultancy, Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses; Amgen: Consultancy, Other: Travel expenses.

scholarly journals Genotype-Phenotype Relationships and Therapeutic Targets in Acute Erythroid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
June Takeda ◽  
Kenichi Yoshida ◽  
Akinori Yoda ◽  
Lee-Yung Shih ◽  
Yasuhito Nannya ◽  
...  
Keyword(s):  
Cell Line ◽  
Board Of Directors ◽  
Research Funding ◽  
Pharmaceutical Research ◽  
Private Company ◽  
Advisory Committees ◽  
Otsuka Pharmaceutical ◽  
Group A ◽  
Genetic Profiles ◽  
Stat Pathway

Background: Acute erythroid leukemia (AEL) is a rare subtype of AML characterized by erythroid predominant proliferation and classified into two subtypes with pure erythroid (PEL) and myeloid/erythroid (MEL) phenotypes. Although gene mutations in AEL have been described in several reports, genotype phenotype correlations are not fully understood with little knowledge about the feasible molecular targets for therapy. Methods: To understand the mechanism of the erythroid dominant phenotype of AEL and identify potential therapeutic targets for AEL, we analyzed a total of 105 AEL cases with the median age of 60 (23-86), using targeted-capture sequencing of commonly mutated genes in myeloid neoplasms, together with 1,279 SNPs for copy number measurements. Among these 105 cases, 13 were also analyzed by RNA sequencing. Genetic profiles of these 105 AEL cases were compared to those of 775 cases with non-erythroid AML (NEL) including 561 cases from The Cancer Genome Atlas and Beat AML study. An immature erythroid cell line (TF1) and three patient-derived xenografts (PDX) established from AEL with JAK2 and/or EPOR amplification. Cell line and samples from patients were inoculated into immune-deficient mice and tested for their response to JAK1/2 inhibitor. Results: According to unique genetic alterations, AEL was classified into 4 subgroups (A-D). Characterized by TP53 mutations and complex karyotype, Group A was the most common subtype and showed very poor prognosis. Remarkably, all PEL cases were categorized into Group A. Conspicuously, 80% of PEL cases had amplifications of JAK2 (6/10; 60%), EPOR (7/10;70%), and ERG (6/10;60%) loci on chromosomes 9p, 19q, and 21q, respectively, frequently in combination, although they were rarely seen in NEL cases. All cases in Group B (n=19, 18%), another prevalent form of AEL, had STAG2 mutations and classified in MEL. To further characterize this subgroup, we compared genetic profiles of STAG2-mutated AEL and NEL. Prominently, 70% (14/20) of STAG2-mutated cases in AEL had KMT2A-PTD, whereas it was found only in 8.8% (3/34) of NEL. CEBPA mutations were also more common in AEL (6/21; 29%) than NEL (4/34; 12%). While Group C was characterized by frequent NPM1 mutations, in contrast to the frequent co-mutation of FLT3 in the corresponding subgroup of NPM1-mutated cases in NEL, NPM1-mutated patents in this subgroup lacked FLT3 mutations but had frequent PTPN11 mutations (8/16; 50%), which were much less common in NEL (25/209; 12%). The remaining cases were categorized into Group D, which was enriched for mutations in ASXL1, BCOR, PHF6, U2AF1 and KMT2C. Recurrent loss-of-function mutations in USP9X were unique to this subtype, although USP9X mutations have been reported in ALL with upregulation of JAK-STAT pathway. In RNA sequencing analysis, AEL cases exhibited gene expression profiles implicated in an upregulated STAT5 signaling pathway, which was seen not only those cases with JAK2 or EPOR amplification, but also those without, suggesting that aberrantly upregulated STAT5 activation might represent a common defect in AEL. Based on this finding, we evaluated the effect of a JAK inhibitior, ruxolitinib, on an AEL-derived cell line and three PDX models established from AEL having TP53 mutations and JAK2 and EPOR mutation/amplification. Of interest, ruxolitinib significantly suppressed cell growth and prolonged overall survival in mice engrafted with TF1 and 2 PDX models with STAT5 downregulation, although the other model was resistant to JAK2 inhibition with persistent STAT5 activation. Conclusion: AEL is a heterogeneous group of AML, of which PEL is characterized by frequent amplifications/mutations in JAK2, EPOR and/or ERG. Frequent involvement of EPOR/JAK/STAT pathway is a common feature of AEL, in which a role of JAK inhibition was suggested. Disclosures Yoda: Chordia Therapeutics Inc.: Research Funding. Shih:Novartis: Research Funding; Celgene: Research Funding; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ishiyama:Alexion: Research Funding; Novartis: Honoraria. Miyazaki:Astellas Pharma Inc.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; NIPPON SHINYAKU CO.,LTD.: Honoraria; Celgene: Honoraria; Otsuka Pharmaceutical: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma KK: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Takaori-Kondo:Celgene: Honoraria, Research Funding; Ono Pharmaceutical: Research Funding; Thyas Co. Ltd.: Research Funding; Takeda: Research Funding; CHUGAI: Research Funding; OHARA Pharmaceutical: Research Funding; Sanofi: Research Funding; Novartis Pharma: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Research Funding; Otsuka Pharmaceutical: Research Funding; Eisai: Research Funding; Astellas Pharma: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Nippon Shinyaku: Research Funding; MSD: Honoraria. Kataoka:Asahi Genomics: Current equity holder in private company; Otsuka Pharmaceutical: Research Funding; Takeda Pharmaceutical Company: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding. Usuki:Alexion: Research Funding, Speakers Bureau; Apellis: Research Funding; Novartis: Research Funding, Speakers Bureau; Chugai: Research Funding. Maciejewski:Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau. Ganser:Novartis: Consultancy; Celgene: Consultancy. Thol:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ogawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Eisai Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: Ruxolitinib is used for drug efficacy test using patient-derived xenografts established from acute erythroid leukemia.

scholarly journals Loss of LKB1/STK11 Facilitates Leukemic Progression of the Myeloproliferative Neoplasms

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 1-1
Author(s):  
Christian Marinaccio ◽  
Praveen K Suraneni ◽  
Hamza Celik ◽  
Andrew Volk ◽  
Jeremy Q. Wen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Private Company ◽  
Blast Phase ◽  
Advisory Committees ◽  
Self Renewal ◽  
Stat Signaling

Nearly 20% of patients with myelofibrosis progress to blast phase disease; an aggressive form of acute myeloid leukemia. Although previous studies have implicated loss of TP53 or JARID2 in progression, by and large the genetic events that lead to conversion to blast phase remain unknown. To identify genes whose loss drives progression, we performed a focused CRISPR/Cas9 screen in which murine Jak2V617F bone marrow cells expressing Cas9 were transduced with two separate sgRNA libraries of known tumor suppressor genes and subjected to colony replating assays. Transduction of one of the two libraries led to serial replating and enhanced self-renewal of the Jak2V617F cells. Subsequent DNA sequencing revealed enrichment of all four guides targeting STK11, the gene that encodes LKB1 which regulates a number of key cellular pathways including energy utilization by activation of AMPK. To confirm that loss of Stk11 is the event that leads to increased clonogenicity, we collected cells from Jak2V617F/Vav-Cre+ and control Vav-Cre+ mice and induced Stk11 knockout by electroporating Cas9-Stk11 sgRNA ribonucleoprotein complexes. Consistent with the screening results, only Jak2V617F Vav-Cre+ cells with Cas9-Stk11 sgRNA showed serial replating. To determine whether Stk11 is required for growth of cells with a different driver of enhanced JAK/STAT signaling, we doubly transduced Stk11 homozygous floxed bone marrow cells with MPLW515L-mCherry and Cre-GFP to delete Stk11. As expected, cells with both MPLW515L and Cre recombinase showed enhanced self-renewal, while singly infected control cells failed to replate. These results demonstrate that activation of JAK/STAT signaling can overcome the requirement for Stk11 in normal hematopoiesis and suggest that STK11 loss may be a strong driver of malignant transformation in combination with enhanced JAK-STAT signaling. We next investigated the mechanism by which loss of STK11 cooperates with enhanced JAK/STAT signaling to promote leukemia. RNA-sequencing of wild-type, Stk11+/+/ MPLW515L, and Stk11-/-/MPLW515L hematopoietic cells revealed enrichment of a number of pathways related to hypoxia, oxidative phosphorylation and mitochondrial translation in cells lacking LKB1. Western blot assays confirmed activation of mTOR signaling as well as HIF1a stabilization and pathway activation, both of which have been reported to lie downstream of LKB1 loss. We also performed a number of studies to determine the relevance of reduced LKB1 expression to leukemic progression. First, we induced deletion of Stk11 in mice that were transplanted with HSPCs expressing MPLW515L after development of the MPN phenotype. Loss of Stk11 caused a rapid lethality that was associated with enhanced bone marrow fibrosis and osteosclerosis. We also observed accumulation of leukemic blasts in small clusters consistent with AML transformation arising in the spent phase MPN. Additionally, we deleted STK11 by CRISPR/Cas9 in primary MPN patient samples and monitored their engraftment in immunocompromised mice. We observed enhanced engraftment and increased reticulin fibrosis and osteosclerosis in mice that received the STK11 edited cells compared to those with non-targeted sgRNA. Third, we compared the expression of STK11 in paired blast and chronic phase myelofibrosis patient samples by RT-PCR. Consistent with the hypothesis that loss of STK11 facilitates leukemia, we found that its expression was decreased by more than 50% in five of seven paired post-MPN AML patient samples, with two having STK11 levels below 20%. We further validated downregulation of LKB1 by immunohistochemistry on paired chronic and blast phase MPN specimens and observed little staining in the blast phase specimens. Finally, to further show that the mechanism of in vitro enhanced self-renewal is related to leukemia progression, we stained the paired marrows for HIF1a and saw a dramatic increase in staining at the AML phase. We also analyzed RNA-seq data of paired chronic versus blast phase MPNs specimens and observed that there is a strong congruence of enriched pathways that are associated with the in vitro mouse HSPC phenotype and the human blast phase progression, such as oxidative phosphorylation and hypoxia. Together, our study demonstrates that loss of LKB1/STK11 promotes transformation of cells with activated JAK/STAT signaling and that STK11 is a prominent candidate tumor suppressor gene in post-MPN AML. Disclosures Gurbuxani: UpToDate: Honoraria. Hoffman:Dompe: Research Funding; Protagonist: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Forbius: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees. Levine:Astellas: Consultancy; Amgen: Honoraria; Gilead: Honoraria; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy. Rampal:Galecto: Consultancy; Incyte: Consultancy, Research Funding; Constellation: Research Funding; Stemline: Consultancy, Research Funding; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; CTI Biopharma: Consultancy; Abbvie: Consultancy; Pharmaessentia: Consultancy; Promedior: Consultancy; Blueprint: Consultancy.

scholarly journals Engineered iPSC-Derived NK Cells Expressing Recombinant CD64 for Enhanced ADCC

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Kate Dixon ◽  
Robert Hullsiek ◽  
Kristin Snyder ◽  
Zachary Davis ◽  
Melissa Khaw ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Target Cells ◽  
Advisory Committees ◽  
High Affinity ◽  
Adcc Assay

Natural killer (NK) cells are innate cytotoxic lymphocytes. They target malignant cells via non-clonotypic receptors to induce natural cytotoxicity and also recognize tumor-bound antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC). While ADCC by NK cells is a key mechanism of several clinically successful therapeutic monoclonal antibodies (mAbs), most patients exhibit or acquire resistance to mAb therapies. ADCC by human NK cells is exclusively mediated by the IgG Fc receptor, CD16A (FcγRIIIA). Studies have demonstrated that increasing the binding affinity between CD16A and therapeutic mAbs can augment their clinical efficacy. Given the exquisite specificity and diverse antigen detection of anti-tumor mAbs, we are interested in enhancing the ADCC potency of NK cell-based therapies for various malignancies. CD64 is the only high affinity FcγR family member and binds to the same IgG isotypes as CD16A (IgG1 and IgG3) but with > 30-fold higher affinity. CD64 (FcγRI) is normally expressed by certain myeloid cells but not by NK cells. We generated a recombinant version of this receptor consisting of the extracellular region of CD64 and the transmembrane and intracellular regions of human CD16A, referred to as CD64/16A (figure 1A). An important feature of CD64/16A is that due to its high affinity state, soluble monomeric anti-tumor mAbs can be pre-adsorbed to engineered NK cells expressing the recombinant FcγR, and these pre-absorbed mAbs can be switched or mixed for universal tumor antigen targeting (figure 1B). The engineered NK cells used in our study were derived from genetically edited and clonally derived induced pluripotent stem cells (iPSCs) through a series of stepwise differentiation stages (figure 2). Engineered iPSC-derived NK (iNK) cells can be produced in a uniform and clinically scalable manner (figure 2). In Figure 3, using an in vitro Delfia® ADCC assay, we show that iNK-CD64/16A cells mediated ADCC against SKOV3 cells, an ovarian adenocarcinoma cell line, in the presence of the anti-HER2 therapeutic mAb trastuzumab (Herceptin) or anti-EGFR1 therapeutic mAb cetuximab (Erbitux), when either added to the assay or pre-adsorbed to the iNK cells (figure 3). Considering the high affinity state of CD64, we examined the effects of free IgG in human serum on ADCC by iNK-CD64/16A cells. Using an IncuCyte® Live Cell Analysis System, ADCC was evaluated in the presence or absence of 5% human AB serum, in which free IgG was approximately 50-fold higher than the IgG saturation level of the CD64/16A receptors on iNK cells (data not shown). Despite the high levels of excess free IgG, iNK-CD64/16A cells mediated efficient ADCC when Herceptin was either added to the assay or pre-adsorbed to the cells (figure 4). ADCC assays were also performed with Raji cells, a Burkitt lymphoma cell line, as target cells and the therapeutic mAb rituximab (Rituxan). iNK-CD64/16A cells were added with or without pre-adsorbed Rituxan and the assay was performed in 10% AB serum. Again, iNK-CD64/16A cells mediated effective target cell killing in the presence of serum IgG (figure 5), demonstrating that saturating levels of free IgG did not prevent ADCC. To determine if we can further optimize the function of recombinant CD64, we engineered CD64 with the transmembrane regions of CD16A or NKG2D and signaling/co-signaling domain from CD28, 2B4 (CD244), 4-1BB (CD137), and CD3ζ (figure 6). CD64/16A signals by non-covalent association with the immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling adapters CD3ζ and FcRγ found in the cell membrane, whereas the other recombinant CD64 constructs use ITAM and non-ITAM regions to mediate their signaling. The various recombinant CD64 constructs were initially expressed in NK92 cells (lacks expression of endogenous FcγRs) (figure 7). Using the Delfia® ADCC assay system, we examined the function of each recombinant CD64 construct and found all combinations are able to effectively induce ADCC (figure 8). We are in the process of generating iNK cells with these constructs and testing their ability to kill hematologic and solid tumors in vitro and in vivo. Our goal is to utilize this docking approach to pre-absorb mAbs to iNK cells for adoptive cell therapy. The mAbs would thus provide tumor-targeting elements that could be exchanged as a means of preventing tumor cell escape by selectively and easily altering NK cell specificity for tumor antigens. Figure Disclosures Lee: Fate Therapeutics, Inc.: Current Employment. Chu:Fate Therapeutics: Current Employment. Abujarour:Fate Therapeutics, Inc: Current Employment. Dinella:Fate Therapeutics: Current Employment. Rogers:Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Miller:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Walcheck:Fate Therapeutics: Consultancy, Research Funding.

scholarly journals Establishing a Novel Pipeline That Combines in-Silico Prediction with in-Vitro and Ex-Vivo Validation to Discover Secondary Drug Combinations Against Relapsed and/or Refractory Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1615-1615
Author(s):  
Sayak Chakravarti ◽  
Suman Mazumder ◽  
Harish Kumar ◽  
Neeraj Sharma ◽  
Ujjal Kumar Mukherjee ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Board Of Directors ◽  
In Silico ◽  
Research Funding ◽  
Ex Vivo ◽  
Standard Of Care ◽  
In Silico Prediction ◽  
Advisory Committees

Abstract Multiple myeloma (MM) is the second-most common hematological malignancy in the US. MM is an incurable, age-dependent plasma cell neoplasm with a 5-year survival rate of less than 50%. Extensive inter-individual variation in response to standard-of-care drugs like proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs), drug resistance, and dose-limiting toxicities are critical problems for the treatment of MM. Clinical success in anti-myeloma treatment, therefore, warrants continuous development of novel combination therapy strategies with the explicit goal to improve the therapeutic efficacy by concomitantly targeting multiple signaling pathways. Previously, we have reported the development of an in-house computational pipeline called secDrug that applies greedy algorithm-based set-covering computational optimization method followed by a regularization technique to predict secondary drugs that can be repurposed as novel synergistic partners of standard-of-care drugs for the management of refractory/ resistant MM. Top among these secondary drugs (secDrugs) were the HSP90 inhibitor 17-AAG. In this study, we used 17-AAG as a proof of principle to establish a pipeline that integrates our in silico predictions with in vitro and ex vivo validation as well as multi-omics technologies to identify, validate, and characterize therapeutic agents that could be used either alone or in combination with standard-of-care drugs for the treatment of R/R MM patients (Figure 1). To screen and validate our in silico prediction results, we performed in vitro cytotoxicity assays using 17-AAG on a panel of human myeloma cell lines (HMCLs; in vitro model systems) that captures a wide range of biological and genetic heterogeneity representing the complexities encountered in clinical settings. These cell lines include HMCLs representing innate sensitive/resistance, >10 pairs of parental and clonally-derived PI- and IMiD-resistant pairs (P vs VR or LenR; representing acquired/emerging resistance/relapse), NRAS mutants which leads to the constitutive activation of oncogenic Ras signaling, and CRISPR-edited HSP90 knockdown cell line. Our results showed that 17-AAG has high synergistic activity in combination with PI in inducing apoptosis even in innate and acquired PI-resistant HMCLs and significantly reduces the effective dose of PI required to achieve IC 50 (Chou-Talalay's Dose Reduction Index or DRI 7±1.4). Moreover, 17-AAG+IMiD showed synergistic cell killing activity in clonally-derived IMiD resistant HMCL. Further, 17-AAG induced cell death was comparable with Hsp90 knockdown as evident from the cytotoxicity assay using PI and 17-AAG in combination in RPMI8226-wild type and RPMI-HSP90AA1 knocked down cell line. Notably, 17-AAG was strikingly effective against the NRAS-mutant cell line indicating an additional niche (NRas mutant myeloma) where 17-AAG could be most effective. Next, we performed RNA sequencing to elucidate the molecular mechanisms behind 17-AAG drug action, drug synergy, 17-AAG-induced cell death. Our gene expression profiling (GEP) followed by Ingenuity Pathway Analysis (IPA) analysis revealed protein ubiquitination, aryl hydrocarbon receptor signalling pathway as the top canonical pathways. 17-AAG induced apoptosis via mitochondrial mediated pathway in myeloma. 17-AAG exerts its cytotoxic effect by activating intrinsic pathway of apoptosis which we further confirmed through the increase in reactive oxygen species generation and decrease in mitochondrial membrane potential. 17-AAG was also effective in reducing the expression of hallmarks of MM such as p65/NF-kB, IRF4, c-Myc. Finally, we performed mass cytometry (CyTOF; Cytometry by time of flight) on primary bone-marrow cells (PMCs) from myeloma patients for further validation of proteomic signatures at the single-cell level. CyTOF analysis confirmed 17-AAG-induced cell death and key changes in MM-specific proteomic markers. 17-AAG treated PMCs showed elevated cleaved caspase levels and down-regulation of IRF4 and phospho-STAT3. GEP and CyTOF results were confirmed using immunoblotting assays. Together, our study demonstrates a unique pipeline for drug repositioning that has the potential to revolutionize clinical decision-making by minimizing the number of drugs required for discovering successful combination chemotherapy regimens against drug-resistant myeloma. Figure 1 Figure 1. Disclosures Kumar: BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Beigene: Consultancy; Oncopeptides: Consultancy; Antengene: Consultancy, Honoraria; Carsgen: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche-Genentech: Consultancy, Research Funding; Bluebird Bio: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding.

scholarly journals Dual Targeting of Ph+ ALL with Dasatinib and ABT-199 (Venetoclax)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1329-1329
Author(s):  
Jessica Leonard ◽  
Joelle Rowley ◽  
Brandon Hayes-Lattin ◽  
Jeffrey W. Tyner ◽  
Marc Loriaux ◽  
...  
Keyword(s):  
Older Adults ◽  
Cell Line ◽  
Board Of Directors ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Annexin V ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Introduction: Treatment of adult Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) remains a challenge. While the addition of the targeted tyrosine kinase inhibitors (TKI) to standard cytotoxic therapy has greatly improved upfront treatment, treatment related mortality in older adults remains high. A novel induction regimen combines the targeted dual Abl/Src TKI Dasatinib (Sprycel, BMS) with a corticosteroid. After the first 21 days of induction the corticosteroids are tapered due to significant toxicities, particularly in older adults. Unfortunately, remaining on TKI monotherapy renders patients susceptible to the development of TKI resistance and thus identifying targeted agents that could enhance the activity of TKIs is urgently needed. Recently a novel and selective inhibitor of BCL-2, ABT-199 (Venetoclax, AbbVie) has shown impressive activity against other lymphoid malignancies including CLL and NHL. Here we describe the pre-clinical and in vivo efficacy of ABT-199 in combination with dasatinib in Ph+ ALL and propose its potential use in future clinical trials. Methods: Drug efficacy in vitro was determined using the Ph+ ALL cell line SupB15, primary Ph+ ALL sample (12-149), the dasatinib sensitive Pre-B ALL cell line RCH and the CML cell line K562. Cells were treated with dasatinib, ABT199 or in combination for 72 hours. Cell viability was assessed with the colorimetric MTS assay and apoptosis was assessed with annexin V staining. Expression of the BCL family proteins BCL-2 and MCL-1 were assessed via immunoblot. Immunodeficient NSG mice were injected with 12-149, then one week later treated with vehicle, 5 mg/kg dasatinib, 5 mg/kg ABT-199, or the combination daily for 5 days each week. Peripheral blood was obtained every 1-2 weeks to assess for engraftment as defined by the presence of >10% human CD45+ cells in the peripheral blood. Once engrafted, mice were euthanized and examined. Mononuclear cells were extracted and assessed for BCL2 and MCL1 expression. Statistical methods were performed using Calcusyn and PRISM. Results: Susceptibility to BCL2 inhibition: Of the dasatinib sensitive cells tested, SupB15 and 12-149 cells were susceptible to ABT-199 while RCH and K562 cells were not. The ALL cells expressed BCL-2 while the CML cell line expressed BCLx. SupB15 expressed low levels of the antiapoptotic protein MCL1 while RCH cells had relatively higher levels. siRNA of MCL-1 rendered the RCH cells sensitive to inhibition by ABT-199. In SupB15 cells, treatment with ABT-199 alone led to upregulation of MCL-1 at 24h which was prevented by the combination of dasatinib + ABT199. Synergy in Ph+ ALL: The calculated IC50 of dasatinib and ABT199 in SupB15 were 8.8nM and 5.9nM, respectively. The IC50 of equimolar combination was 0.42nM, and synergistic with combination index (CI) values between 0.15 and 0.49. Primary Ph+ ALL xenograft cells showed a similar pattern of synergy to the dasatinib + ABT199 combination. Combination treatment also greatly increased apoptosis as measured by Annexin V staining. Xenograft Studies: Animals were treated with a ten-fold lower dose of dasatinib and ABT199 from prior published data. There was no significant difference in time to engraftment or disease burden between vehicle or single agent ABT-199. In contrast, less than one half of the animals treated with dasatinib engrafted by 90 days while none of the animals treated with both dasatinib and ABT-199 engrafted. Most intriguing was the decrease in disease burden as measured by splenic size in the combination group compared to all other groups (P<0.0001, one-way ANOVA). Analysis of BCL-2 family proteins from mononuclear cells isolated from untreated animals confirmed upregulation of BCL-2 and relatively low levels of MCL-1. Animals treated with ABT-199 had greatly upregulated levels of MCL-1, while those treated with dasatinib or the combination did not. Conclusions: The combination of ABT-199 with dasatinib synergistically targets Ph+ ALL cells both in vitro and in vivo, laying the foundation for further evaluation in vivo for adult Ph+ ALL. As demonstrated by others, malignancies that are particularly susceptible to BCL targeting are those which display high BCL-2 expression and a low MCL-1: BCL-2 ratio. Combined targeted therapies may offer the potential for greater and longer responses without the morbidity associated with cytotoxic chemotherapy, particularly in older adults. Disclosures Tyner: Aptose Biosciences: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Array Biopharma: Research Funding; Constellation Pharmaceuticals: Research Funding. Druker:Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; Novartis Pharmaceuticals: Research Funding; Sage Bionetworks: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Henry Stewart Talks: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotide Pharmaceuticals: Research Funding; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy.

At-101 Induces Apoptosis Waldenström Macroglobulinemia Cells Resistant to Bortezomib.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2861-2861
Author(s):  
Kasyapa Chitta ◽  
Kiersten M. Miles ◽  
Pushpankur Ghoshal ◽  
Leighton Stein ◽  
Morton Coleman ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Board Of Directors ◽  
Therapeutic Potential ◽  
Cross Resistance ◽  
Advisory Committees ◽  
Cellular Activation ◽  
Potent Inducer ◽  
Resistant Cells

Abstract Abstract 2861 Poster Board II-837 Bcl-2 protein family has the unique capability to balance between the cell survival and death by regulating the expression of its individual members. AT-101 is a BH3 mimetic and a potent inducer of noxa and puma, the natural ligands of BH3 family proteins. It is known to bind and inhibit the anti-apoptotic functions of Bcl-2 family members Bcl-2 and Bcl-XL and Mcl-1. In vitro it has been shown to induce apoptosis in several tumor models systems including multiple myeloma. In this report we investigated the effect of AT-101 on a model cell line, BCWM.1 (a known WM cell line, BCWM.1/WT), representing Waldenström Macroglobulinemia. This disease is characterized by the presence of lymphoplasmacytic cells in the bone marrow and the secretion of IgM monoclonal protein into the serum. Several conventional therapies are available but the disease remains incurable. Therefore there remains a need to develop new therapies for this orphan disease. Recently, bortezomib (a proteasomal inhibitor) has shown promising anti-WM activity with enhanced responses when combined with traditional therapies. But continued treatment with bortezomib result in the development of resistance in the clinic. We developed an in vitro model of bortezomib resistance from BCWM.1 (hereafter referred as BCWM.1/BR). These cells also developed cross resistance to conventional therapies used for WM such as fludarabine and doxorubicin. Biological characterization of this cell line demonstrated Bcl-2 as a potentially important therapeutic target. We therefore assessed the effect of AT-101 to identify preclinically if this could be a potential clinical strategy in future. AT-101 induced a dose and time dependent inhibition in the viability of both BCWM.1/WT as well as BCWM.1/BR cells. Cell death was observed at as low as 1uM concentration of AT-101 and at 10uM a maximum of 50-70% death was observed by 24h. While BCWM.1/WT cells showed a significant death at 12h, treatment with AT-101 induced cell death in BCWM.1/BR cells as early as 6h. These results indicate that AT-101 induced a potent and quick inhibition in viability in BCWM.1/BR cells as compared to their parental wild type cells. Investigation into the mechanism of cell death showed that AT-101 induced apoptosis in a mitochondrial dependent pathway in these cells. A comparative analysis of the signal transduction pathways operated in BCWM.1/WT and BCWM.1/BR cells showed that many of the cellular activation and survival pathways such as AKT, ERK1/2 that are present in BCWM.1 cells are inhibited in the resistant cells. Interestingly, BCWM.1/BR cells expressed a fivefold increase in the Bcl-2 protein as compared to BCWM.1/WT cells suggesting a Bcl-2 dependent survival of these cells in the absence of other cellular activation and survival signals. Increased susceptibility of BCWM.1/BR cells to AT-101 thus can be understood to be a direct consequence of an increased expression of Bcl-2 and a dependence of the resistant cells on Bcl-2 family of anti-apoptotic proteins for their survival. Results presented in this report suggest that AT-101 has a unique therapeutic potential against Waldenström Macroglobulinemia that is independent of resistance to bortezomib. These observations highlight bcl-2 as a potential target, and AT-101 as possible therapeutic avenue for WM patients. Disclosures: Chanan-Khan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immunogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals Older Age and Increased Neutrophil-to-Lymphocyte Ratio (NLR) Are Predictors of Mortality in a Multiethnic Urban Cohort of Hematologic Neoplasms and COVID-19 Patients

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-35
Author(s):  
Angelica D'Aiello ◽  
Sumaira Zareef ◽  
Kith Pradhan ◽  
Amanda Lombardo ◽  
Fariha Khatun ◽  
...  
Keyword(s):  
Cancer Treatment ◽  
Board Of Directors ◽  
Median Time ◽  
Lymphocyte Count ◽  
Research Funding ◽  
Hematologic Neoplasms ◽  
Private Company ◽  
Advisory Committees ◽  
Treatment Status ◽  
Race Ethnicity

Introduction: We sought to compare outcomes among patients with hematologic neoplasms diagnosed with COVID-19 infection in a multiethnic urban academic medical center. Methods: A retrospective analysis of patients with hematologic neoplasms diagnosed with COVID-19 from March 17th to June 8th2020 was conducted. Subjects included were censored at last point of contact. Variables collected included age, gender, race/ethnicity, hematologic diagnosis, cancer treatment status, baseline and follow-up COVID-19 testing, neutrophil count, and lymphocyte count at time of diagnosis. Associations between hematologic diagnosis, cancer treatment status, age, gender, race/ethnicity, neutrophil-to-lymphocyte ratio (NLR), and overall survival (OS) were assessed using the Kaplan-Meier method with logrank test. Results: A total of 102 subjects with hematologic neoplasms and COVID-19 infection treated in Montefiore Health system were identified (Table 1). Thirty-nine (38%) subjects were undergoing active treatment, including 17 (16%) receiving conventional chemotherapy agents, 12 (12%) targeted therapy, and 10 (10%) combination therapy. Of those subjects, twenty (50%) experienced delay or discontinuation of treatment due to COVID-19 infection. Four subjects (4%) showed persistent infection by PCR at median duration of 25.1 days after initial diagnosis. Ten subjects (9.8%) showed clearance of the virus by PCR with median time-to-clearance of 51.8 days. Of 9 subjects with serologic testing, 8 tested positive for COVID-19 IgG antibody at median time of 62 days after initial COVID-19 diagnosis. Forty-seven (47%) subjects expired as a result of COVID-19 disease at the time of analysis. Disease type, treatment status, race/ethnicity, age, and gender showed no significant association with mortality. Patients older than 70 had worse outcomes than the younger population (p = 0.0082). Median neutrophil and lymphocyte count at time of diagnosis was 4500 and 900, respectively. NLR greater than 9 was associated with worse survival when compared to NLR less than 9 (p=0.0067). Conclusions: COVID-19 infection has adverse effects on patients with hematological neoplasms. Subjects older than 70 years had a significantly worse prognosis. Notably, subjects actively being treated with chemotherapy did not have worse outcomes than those not being treated in our cohort, supporting the notion than active COVID-19 infection per se should not result in treatment delays. In addition, high NLR correlates with worsened survival, suggesting that this could be a potential prognostic factor for COVID-19 mortality in the hematologic neoplasms population. Disclosures Steidl: Stelexis Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Research Funding; Pieris Pharmaceuticals: Consultancy; Aileron Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Verma:stelexis: Current equity holder in private company; BMS: Consultancy, Research Funding; Medpacto: Research Funding; Janssen: Research Funding; acceleron: Consultancy, Honoraria.

The Interaction of Bortezomib with P-Gp, MRP-1 and BCRP Drug Transporters: Implications for Therapeutic Applications of Bortezomib in Advanced Multiple Myeloma and Other Neoplasias.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1729-1729
Author(s):  
Melissa G Ooi ◽  
Robert O'Connor ◽  
Jana Jakubikova ◽  
Justine Meiller ◽  
Steffen Klippel ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Significant Activity ◽  
Cancer Resistance ◽  
Advisory Committees ◽  
Bortezomib Treatment

Abstract Abstract 1729 Poster Board I-755 Background Multidrug transporters are energy-dependent transmembrane proteins which can efflux a broad range of anticancer drugs and thereby play a role in resistance to the actions of substrate agents. Classically, three transporters, p-glycoprotein (Pgp; MDR-1; ABCB1), multidrug resistant protein-1 (MRP-1; ABCC1) and breast cancer resistance protein (BCRP; MXR; ABCG2), have been found to have the broadest substrate specificity and a strong correlation with drug resistance in vitro and in vivo in many models and forms of cancer. We have sought to characterize the interaction of bortezomib with these transporters and thereby explore the potential for these agents to play a role in resistance. Bortezomib is a novel proteosome inhibitor with significant activity in multiple myeloma, although subsets of patients remain refractory to the activity of the drug. Hence, better characterization of the interactions of this drug with classical resistance mechanisms may identify improved treatment applications. Methods and Results We investigated the role of these transporters by using isogenic cell line models which are resistant due to overexpression of a particular transporter: DLKP lung cancer cell line that overexpresses MRP-1; DLKP-A which overexpresses Pgp; and DLKP-SQ-Mitox which overexpresses BCRP. DLKP-A cells exhibited a 4.6-fold decrease in responsiveness to bortezomib compared to parental DLKP cells. In DLKP-SQ-Mitox, bortezomib-induced cytotoxicity was comparable to DLKP. When bortezomib was combined with elacridar, a Pgp and BCRP inhibitor, significant synergy was evident in DLKP-A (100% viable cells with single agent treatment versus 11% with the combination), but not DLKP-SQ-Mitox. Sulindac, an MRP-1 inhibitor, combined with bortezomib failed to produce any synergy in MRP-1 positive DLKP cells. Conversely, combination assays of Pgp substrate cytotoxics such as doxorubicin with Bortezomib were largely additive in nature. This indicates that bortezomib has little, if any, direct Pgp inhibitory activity, as combinations of a traditional Pgp inhibitor (such as elacridar) and doxorubicin would show marked synergy rather than just an additive effect in Pgp positive cells. To further characterize the extent of this interaction with Pgp, we conducted cytotoxicity assays in cell lines with varying levels of Pgp overexpression. NCI/Adr-res (ovarian cancer, high Pgp overexpression), RPMI-Dox40 (multiple myeloma, moderate Pgp overexpression) and A549-taxol (lung cancer, low Pgp overexpression). The combination of bortezomib and elacridar that produced the most synergy was in cell lines expressing moderate to high levels of Pgp expression. Cell lines with lower Pgp expression produced an additive cytotoxicity. We next examined whether bortezomib had any direct effect on Pgp expression. In RPMI-Dox40 cells, Pgp expression is reduced in a time-dependent manner with bortezomib treatment. Conclusions Our studies therefore show that bortezomib is a substrate for Pgp but not the other drug efflux pumps. In tumor cells expressing high levels of Pgp, the efficacy of bortezomib is synergistically enhanced by combinations with a Pgp inhibitor, while bortezomib treatment itself can reduce the expression of Pgp. This study suggests that in the subset of patients with advanced multiple myeloma or solid tumors which express high levels of Pgp, inhibition of its function could contribute to enhanced responsiveness to bortezomib. Disclosures Richardson: millenium: 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, speakers bureau up to 7/1/09; MLNM: speakers bureau up to 7/1/09. Mitsiades:Millennium Pharmaceuticals : Consultancy, Honoraria; Novartis Pharmaceuticals : Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: licensing royalties ; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono : Research Funding; Sunesis Pharmaceuticals: Research Funding. Anderson:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Biotest AG: Consultancy, Research Funding.

Response and Outcomes to Nilotinib at 24 Months in Imatinib-Resistant Chronic Myeloid Leukemia Patients in Chronic Phase (CML-CP) and Accelerated Phase (CML-AP) with and without BCR-ABL Mutations.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1130-1130 ◽  
Author(s):  
Jerald P. Radich ◽  
Giovanni Martinelli ◽  
Andreas Hochhaus ◽  
Enrico Gottardi ◽  
Simona Soverini ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Median Time ◽  
Research Funding ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Cytogenetic Response ◽  
Molecular Response ◽  
Advisory Committees ◽  
Imatinib Resistant

Abstract Abstract 1130 Poster Board I-152 Background Nilotinib is a selective and potent BCR-ABL inhibitor, with in vitro activity against most BCR-ABL mutants (excluding T315I) indicated for the treatment of patients with Philadelphia chromosome positive (Ph+) CML in CPor AP resistant or -intolerant to prior therapy, including imatinib. In a previous analysis of nilotinib in patients with BCR-ABL mutations, mutations occurring at three specific amino acid residues (E255K/V, Y253H, and F359C/V) were shown to be associated with less favorable response to nilotinib. The current analysis is based on mature data with a minimum follow-up of 24-months for all patients. Outcomes of patients at 24 months were analyzed by mutation type. Methods Imatinib-resistant CML-CP (n = 200) and CML-AP (n = 93) patients were subdivided into the following mutational subsets: no mutation, sensitive mutations (including mutations with unknown in vitro IC50). or E255K/V, Y253H, or F359C/V mutations at baseline. Patients with mutations of unknown in vitro sensitivity were classified as sensitive in this analysis based on a previous finding that patients with these mutations responded similarly to nilotinib as patients with sensitive mutation. Patients with baseline T315I mutations were excluded from this analysis. Patient groups were analyzed for kinetics and durability of cytogenetic and molecular response to nilotinib, as well as event-free survival (EFS), defined as loss of hematologic or cytogenetic response, progression to AP/BC, discontinuation due to disease progression, or death, and overall survival (OS). Results In CML-CP and -AP patients with no mutation, sensitive mutations, or E255K/V, Y253H, or F359C/V mutations, hematologic, cytogenetic and molecular responses are provided in the Table. Overall, patients with no mutations responded similarly to patients with sensitive mutations, whereas patients with E255K/V, Y253H, or F359C/V mutations had less favorable responses. This correlation was observed in both CML-CP and CML-AP patients, respectively. Median time to CCyR was 3.3 months (range, 1.0–26.7) for CML-CP patients with no mutations, and 5.6 months (range, 0.9–22.1) for patients with sensitive mutations. At 24 months, CCyR was maintained in 74% of CML-CP patients with no mutation and in 84% of patients with sensitive mutations. One patient with CML-CP and an E255K mutation achieved CCyR at 25 months and maintained until last assessment at 30 months. Median time to MMR was similar at 5.6 months (range, 0.9–25.8) for CML-CP patients with no mutations and 5.6 months (range, 2.7–22.1) for patients with sensitive mutations. No patient with a less sensitive mutation achieved MMR. Median EFS and 24-month estimated OS rate are provided in the Table. Conclusions Imatinib-resistant CML-CP and CML-AP patients treated with nilotinib therapy with BCR-ABL mutations (excluding E255K/V, Y253H, or F359C/V) achieved rapid and durable cytogenetic responses, and estimated EFS and OS at 24 months similar to that of patients with no mutations, respectively. Patients with E255K/V, Y253H, or F359C/V mutations had lower and less-durable responses and shorter EFS than patients with sensitive mutations. Alternative therapies may be considered for patients with these uncommon mutations (E255K/V, Y253H, and F359C/V). Disclosures Radich: Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Novartis: Research Funding. Branford:Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Shou:Novartis: Employment. Haque:Novartis: Employment. Woodman:Novartis: Employment. Kantarjian:Novartis: Research Funding. Hughes:Bristol-Myers Squibb: Advisor, Honoraria, Research Funding; Novartis: Advisor, Honoraria, Research Funding. Kim:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Wyeth: Research Funding. Saglio:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau.

CYC065, a Potent Derivative of Seliciclib Is Active In Multiple Myeloma In Preclinical Studies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2999-2999 ◽  
Author(s):  
Samantha Pozzi ◽  
Diana Cirstea ◽  
Loredana Santo ◽  
Doris M Nabikejje ◽  
Kishan Patel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Preliminary Data ◽  
Research Funding ◽  
Preclinical Studies ◽  
Dependent Manner ◽  
Advisory Committees

Abstract Abstract 2999 Multiple myeloma (MM) is a treatable but incurable hematological malignancy and novel targeted therapies are under investigation. MM is characterized by dysregulation of the cell cycle, consequent to the overexpression of cyclins and their related kinases, the cyclins dependent kinases (CDK), a group of Ser/Thr proteine kinases. CDKs represent a promising therapeutic target, and inhibitors have been developed for anticancer treatment. We have previously studied seliciclib in the context of MM. CYC065, a second generation CDK inhibitor is the more potent derivative of seliciclib. It is mainly active on CDK 2, 5 and 9, involved in progression of the cell cycle and protein transcription. It has already shown promising results in preclinical studies in breast cancer and acute leukemia. We tested CYC065 in in vitro experiments in MM. Our preliminary data in 7 MM cell lines showed cytotoxicity of CYC065, both in MM cell lines sensitive as well as resistant to conventional chemotherapy, with an IC50 ranging between 0.06 and 2μ M, at 24 and 48h. Tritiated thymidine uptake assay confirmed the antiproliferative effects of CYC065 in MM, and its ability to overcome the growth advantage conferred by co-culture with bone marrow stromal cells derived from MM patients, and cytokines like interleukin 6 (10ng/ml) and insulin like growth factor-1 (50ng/ml). The anti-proliferative effect was evident both at 24 and 48h, starting at concentrations as low as 0.015μ M. The AnnexinV/PI assay in the MM1.s cell line confirmed CYC065's ability to induce apoptosis in a time dependent manner starting at 9 hours of treatment, at a concentration of 0.125 μ M, inducing 82% of apoptosis after 48h of exposure. Cell cycle analysis in the same MM1.s cell line showed an increase of subG1 phase, starting at 9 hours of treatment, at 0.125 μ M of CYC065. Preliminary results of western blot analysis confirmed the apoptotic effect of CYC065 in the MM1s cell line, highlighted by the cleavage of caspase 3, 8, 9 and PARP. The compound was tested in primary CD138+ cells isolated from three refractory MM patients, confirming its efficacy at 0.125 μ M, both at 24 and 48h. Comparative analysis in PBMCs from normal donors, for the evaluation of the drug toxicity is ongoing and will be presented. In conclusion our preliminary data confirm the efficacy of CYC065 in MM cell lines and primary MM cells, at nanomolar concentrations. Ongoing mechanistic and in vivo studies will delineate its role in the now increasing spectrum of CDK inhibitors in MM and better define its potential for clinical development in MM. Disclosures: Green: Cyclacel: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Acetylon: Research Funding.

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