Mechanistic Basis and Efficacy of Targeting β-Catenin-TCF7L2-JMJD6-MYC Axis to Overcome Resistance to BET Inhibitors

Hematopoietic progenitor cells of myeloproliferative neoplasms with myelofibrosis (MPN-MF) exhibit constitutive activation of JAK-STAT5/3 and NFkB signaling. Transformation of MPN-MF to AML (post-MPN sAML) occurs in up to 15% of patients with MPN-MF. Standard induction anti-AML chemotherapy and the JAK1 & 2 inhibitor (JAKi) ruxolitinib are ineffective in post-MPN sAML. BET protein BRD4 is a non-oncogene addiction target in AML, and treatment with acetyl-lysine mimetic BET protein inhibitor (BETi) disrupts binding of BRD4 to acetylated chromatin and transcription factors (TFs). This attenuates transcription of super-enhancer regulated oncogenes, including MYC, Bcl-xL, PIM1 and CDK4/6, inhibiting growth and survival of post-MPN sAML blasts. BETi treatment also inhibits binding of BRD4 to acetylated RELA (NFkB-p65), inhibiting its transcriptional activity and attenuating levels of its target cytokines. However, BETi treatment induces BRD4, potentially reducing BETi activity in repressing oncogenes. Preclinical and clinical studies have demonstrated that innate or adaptive BETi-resistance is common in sAML cells. To model BETi-resistance, we repeatedly exposed (10 times) secondary (s) AML SET2 and HEL92.1.7 (HEL) cells to 1.0 µM of the BETi OTX015 for 48 hours followed by full recovery, thus generating BETi persister-resistant (BETi-P/R) SET2-P/R and HEL-P/R cells. These cells showed > 10-fold resistance to OTX015 and cross-resistance to other BETis. Compared to the parental controls, BETi-P/R cells lacked additional genetic alterations or altered levels of TRIM33, SPOP, DUB3 or phosphorylated BRD4 (previously described mechanisms of BETi-resistance). However, ATAC-Seq and ChIP-Seq (H3K27Ac mark) analyses demonstrated that, as compared to their parental controls, BETi-P/R cells showed gain of peaks and active enhancers with enrichment of STAT5, MYC, PU.1 and GATA2 binding sites. Newly gained peaks were in the enhancers of JAK1/2, RUNX1, PU.1, MYC and BCL2L1. RNA-Seq determined mRNA level alterations, included induction of gene-sets involving MYC/MAX, STAT5, NFkB and TCF7L2 targets. QPCR and Western analyses confirmed increase in the mRNA and protein levels of TCF7L2, JMJD6, c-Myc, Survivin and PIM1 in HEL-P/R over HEL92.1.7 cells. Expression of the arginine demethylase JMJD6, recruited by BRD4 to regulate enhancer-mediated transcriptional pause-release, was also increased. This was associated with increased expression of the nuclear β-catenin-TCF7L2 targets, including Cyclin D1, TERT, survivin, c-Myc and PU.1. Patient-derived human AML blasts that exhibited innate resistance ex vivo to BETi, also demonstrated increased expression of TCF7L2, JMJD6 and c-Myc. We next probed the mechanistic role of the β-catenin-JMJD6-TCF7L2-MYC axis in conferring BETi-resistance. CRISPR-Cas9-mediated knockout of TCF7L2 or JMJD6 significantly reversed BETi-resistance in BETi-P/R sAML cells (p < 0.001). Conversely, ectopic overexpression of TCF7L2 or JMJD6 significantly conferred BETi-persister-resistance in HEL and SET2 cells (p < 0.001). Notably, confocal microscopy demonstrated increased binding of β-catenin with TBL1 and TCF7L2 in the nucleus of BETi-P/R sAML cells. BC2059, which disrupts binding of nuclear β-catenin with TBL1 and TCF7L2, depleted β-catenin levels and exerted similar lethality in BETi-P/R sAML and control sAML cells. shRNA-mediated knockdown of BRD4 and treatment with BRD4-PROTAC (proteolysis-targeting chimera) ARV-771 (Arvinas, Inc.) that degrades BRD4/3/2, also induced similar levels of apoptosis in BETi-P/R and control sAML cells. Co-treatment with ARV-771 and BC2059 synergistically induced lethality in BETi-P/R sAML cells as well as in patient-derived, CD34+ sAML BPCs (combination indices < 1.0). This was associated with marked attenuation of c-Myc, TCF4, Survivin, CDK6, PIM1 and Bcl-xL levels. Also, compared to each agent alone, in vivo treatment with ARV-771 (30 mg/kg SQ daily x 5, per week) and BC2059 (30 mg/kg IP BIW per week) for 3 weeks, significantly reduced sAML burden and improved survival of NSG mice engrafted with HEL-P/R cells (p < 0.01). Collectively, these findings underscore that increased levels and activity of β-catenin-TCF7L2-JMJD6-MYC axis is mechanistically responsible for BETi-P/R, and co-targeting with BRD4 degrader and β-catenin-TCF7L2 inhibitor is a promising therapeutic strategy against BETi-P/R sAML BPCs. Disclosures Bhalla: Beta Cat Pharmaceuticals: Consultancy. Verstovsek:Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding.

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Novel BET Protein Degrader-Based Combinations Exert Lethality Against Human AML Resistant to BET Inhibitors Due to Increased Activity of β-Catenin-TCF7L2- MYC Axis

Blood ◽

10.1182/blood-2018-177 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 177-177

Author(s):

Dyana T. Saenz ◽

Warren Fiskus ◽

Taghi Manshouri ◽

David N Saenz ◽

Raffaella Soldi ◽

...

Keyword(s):

Transcription Factor ◽

Progenitor Cells ◽

Board Of Directors ◽

Research Funding ◽

Genetic Alterations ◽

Advisory Committees ◽

Lethal Effects ◽

Bet Inhibitors ◽

Nuclear Levels ◽

And Control

Abstract Bromodomain and extra-terminal protein (BETP) inhibitors (BETis) disrupt the chromatin binding and activity of the BETP BRD4 in facilitating RNA pol II-mediated mRNA transcription, thereby depleting levels of active oncoproteins including c-Myc, CDK6, BCL2, PIM1 and MCL1. BETi treatment also increases protein levels of p21, p27 and HEXIM1, thereby causing growth inhibition and apoptosis of AML blast progenitor cells (BPCs), including post-MPN, secondary AML (sAML) BPCs. Treatment with BETi (e.g., OTX015) has been shown to reduce AML burden and induce clinical remissions. However, BETi-refractory AML develops uniformly. Previous reports utilizing mouse AML models have highlighted that persister-resistance to BETi (BETi-P/R) in AML stem progenitor cells is observed despite BETi treatment and reduction of BRD4 occupancy on the chromatin. This is mediated by re-expression of c-Myc due to transcriptional activity of WNT-β-catenin. In the present studies, we developed human sAML models of BETi-P/R to elucidate the mechanisms and develop targeted therapies against BETi-P/R sAML BPCs. Utilizing human sAML control (parental) SET2 and HEL92.1.7 cells and subjecting them to at least 10 exposures to 1.0 µM of the BETi OTX015 for 48 hours followed by full recovery, we first generated the BETi-P/R SET2-P/R and HEL-P/R cells. These cells were > 10-fold resistant to OTX015 and exhibited cross-resistance to other BETis, including JQ1 and ABBV-075. As compared to the control sAML cells, SET2-P/R and HEL-P/R cells neither exhibited additional genetic alterations by NextGen whole-exome sequencing, nor showed altered levels of TRIM33, SPOP or phosphorylated BRD4 (previously described mechanisms of BETi-resistance). In contrast, compared to the control, SET2-P/R and HEL-P/R cells demonstrated significantly higher nuclear levels and binding of β-catenin to the transcription factor TCF7L2 (TCF4) and TBL1X (TBL1), associated with increased expression of TCF4 targets, including c-Myc, Cyclin D1, TERT and Survivin. ATAC-Seq and ChIP-Seq (H3K27Ac mark) analyses showed significant gain of peaks and active enhancers in HEL-P/R over HEL92.1.7 cells, including enrichment of the STAT5, MYC, PU.1, GATA2 and MYB transcription factor binding sites, as well as newly gained peaks in the enhancers of JAK1/2, RUNX1, PU.1, MYC, BCL2L1 and CTNNB1. RNA-Seq analysis showed significant increase/decrease in mRNA expressions (340/247), with increased expression of gene-sets involving MYC/MAX, STAT5, NFkB and TCF4 targets. QPCR and Western analyses confirmed significant perturbation in gene expressions, with increase in TCF4, c-Myc, Survivin and PIM1 in HEL-P/R over HEL92.1.7 cells. Consistent with the finding that shRNA-mediated knockdown of BRD4 exerted similar lethal effects in BETi-P/R versus control cells, we also discovered that BETP-PROTAC (proteolysis targeting chimera) ARV-771 (Arvinas, Inc.) that degraded BRD4/3/2 was equipotent in inducing apoptosis of BETi-P/R and control sAML cells. Also, consistent with increased nuclear levels and binding (utilizing confocal microscopy) of β-catenin with TBL1 and TCF4 in BETi-P/R sAML BPCs, β-catenin inhibitor BC2059 (Beta-Cat Pharma), which disrupts the binding of nuclear β-catenin with TBL1 and TCF4 and depletes β-catenin levels, exerted similar lethal effects in BETi-P/R sAML and control sAML cells. Consistent with these findings, we also determined that co-treatment with ARV-771 and BC2059 exerted synergistic in vitro lethality against BETi-P/R sAML BPCs (combination indices < 1.0), which was associated with greater reduction in levels of c-Myc, TCF4, Survivin, CDK6, PIM1 and Bcl-xL. Co-treatment with ARV-771 and BC2059 was also synergistically lethal against 12 patient-derived samples of CD34+ sAML BPCs. Notably, compared to treatment with each agent alone or vehicle control, in vivo treatment with ARV-771 (30 mg/kg SQ daily x 5, per week) and BC2059 (30 mg/kg IP BIW per week) for 3 weeks, significantly reduced the sAML burden and improved survival of the NSG mice engrafted with luciferase-transduced HEL-P/R cells (p < 0.01). These findings demonstrate that increased levels and activity of β-catenin-TCF7L2-MYC axis is mechanistically responsible for BETi-P/R, and co-targeting with BETP degrader and β-catenin-TCF4 inhibitor is synergistically lethal against BETi-P/R sAML BPCs. Disclosures Soldi: Beta Cat Pharma: Employment. Bose:Astellas Pharmaceuticals: Research Funding; Celgene Corporation: Honoraria, Research Funding; Blueprint Medicines Corporation: Research Funding; Pfizer, Inc.: Research Funding; Constellation Pharmaceuticals: Research Funding; CTI BioPharma: Research Funding; Incyte Corporation: Honoraria, Research Funding. Kadia:BMS: Research Funding; Takeda: Consultancy; Novartis: Consultancy; Celgene: Research Funding; BMS: Research Funding; Jazz: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy; Celgene: Research Funding; Jazz: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Pfizer: Consultancy, Research Funding; Abbvie: Consultancy; Abbvie: Consultancy. DiNardo:Abbvie: Honoraria; Medimmune: Honoraria; Karyopharm: Honoraria; Celgene: Honoraria; Bayer: Honoraria; Agios: Consultancy. Horrigan:Beta Cat Pharma: Employment. Khoury:Stemline Therapeutics: Research Funding. Verstovsek:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy.

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Pre-Clinical Efficacy of Co-Treatment with KDM1A (LSD1) Inhibitor and Ruxolitinib or BET Inhibitor Against Post-MPN-sAML Blast Progenitor Cells

Blood ◽

10.1182/blood-2019-130783 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1274-1274

Author(s):

Warren Fiskus ◽

Christopher Peter Mill ◽

Vrajesh Karkhanis ◽

Bernardo H Lara ◽

Prithviraj Bose ◽

...

Keyword(s):

Growth Inhibition ◽

Progenitor Cells ◽

Board Of Directors ◽

Research Funding ◽

Amine Oxidase ◽

Genetic Alterations ◽

Advisory Committees ◽

Bet Inhibitor ◽

Oncology Research ◽

Differentiation Block

LSD1 (KDM1A) is an FAD-dependent amine-oxidase that demethylates mono and dimethyl histone H3 lysine 4 (H3K4Me1 and H3K4Me2), which regulates active enhancers and transcription in AML stem/progenitor cells (LSCs). LSD1 is part of the repressor complexes involving HDACs, CoREST or GFI1, mediating transcriptional repression and differentiation block in LSCs that persist in the minimal residual disease (MRD) following attainment of clinical complete remission, leading to relapse and poor outcome in AML. In AML LSCs, genetic alterations and epigenetic dysregulation of enhancers affect levels of myeloid transcriptional regulators, including c-Myc, PU.1, GATA 2 and CEBPα, and their target genes, which are involved in differentiation block in LSCs. Our present studies demonstrate that CRISPR/Cas9-mediated knockout of LSD1 in the AML OCI-AML5 cells significantly increased the permissive H3K4Me2/3-marked chromatin, reduced H3K27Ac occupancy at super-enhancers and enhancers (SEs/Es) (by ChIP-Seq), especially of c-Myc and CDK6, as well as repressed CoREST, c-Myc, CDK6, and c-KIT, while inducing p21, CD11b, and CD86 levels (log2 -fold change by RNA-Seq, and protein expression by Western analyses). This led to significant growth inhibition, differentiation and loss of viability of OCI-AML5 and patient-derived AML blasts (p < 0.01). Similar effects were observed following exposure of OCI-AML5 (96 hours) to tet-inducible shRNA to LSD1. Knock-down of GFI1 by shRNA (by 90%) also inhibited growth and induced differentiation, associated with upregulation of PU.1, p21 and CD11b levels. Treatment with irreversible (INCB059872, 0.25 to 1.0 µM) or reversible (SP2577, 1.0 to 2.0 µM) LSD1 inhibitor (LSD1i) inhibited binding of LSD1 to CoREST, and significantly induced growth inhibition, differentiation and loss of viability (over 96 hours) of the OCI-AML5, post-myeloproliferative neoplasm (post-MPN) sAML SET2 and HEL92.1.7 cells, as well as patient-derived AML and post-MPN sAML blasts (p < 0.01). Co-treatment with INCB059872 and ruxolitinib synergistically induced apoptosis of the post-MPN sAML SET2 and HEL92.1.7 cells and patient-derived CD34+ post-MPN sAML blasts (combination indices < 1.0). Notably, pre-treatment with the LSD1i for 48 hours significantly re-sensitized ruxolitinib-persister/resistant SET2 and HEL92.1.7 cells to ruxolitinib (p < 0.001). We previously reported that treatment with the BET inhibitor (BETi) JQ1 or OTX015 represses SE/E-driven AML-relevant oncogenes including MYC, RUNX1, CDK6, PIM1, and Bcl-xL, while inducing p21 and p27 levels in post-MPN sAML blasts (Leukemia 2017;31:678-687). This was associated with inhibition of colony growth and loss of viability of AML and post-MPN sAML blasts (p < 0.01). Here, we determined that INCB059872 treatment induced similar levels of lethality in BETi-sensitive or BETi-persister/resistant AML and post-MPN sAML cells. Since BETi treatment also depleted LSD1 protein levels, co-treatment with the BETi OTX015 and LSD1i INCB059872 or SP2577 induced synergistic lethality in AML and post-MPN sAML blasts (combination indices < 1.0). Co-treatment with INCB059872 (1.5 mg/kg) and OTX015 (50 mg/kg) both orally for 21 days, compared to each agent alone or vehicle control, significantly reduced the sAML burden and improved survival of immune-depleted mice engrafted with HEL92.1.7 or HEL92.1.7/OTX015-resistant-GFP/Luc sAML xenografts (p < 0.01). Collectively, these findings strongly support further in vivo testing and pre-clinical development of LSD1i-based combinations with ruxolitinib against post-MPN sAML and with BETi against AML or post-MPN sAML cells. Disclosures Bose: CTI BioPharma: Research Funding; Astellas: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Constellation: Research Funding; Incyte Corporation: Consultancy, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding; Blueprint Medicine Corporation: Consultancy, Research Funding; Kartos: Consultancy, Research Funding; Pfizer: Research Funding. Kadia:Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Bioline RX: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees. Bhalla:Beta Cat Pharmaceuticals: Consultancy. Khoury:Stemline Therapeutics: Research Funding; Angle: Research Funding; Kiromic: Research Funding. Verstovsek:Ital Pharma: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Incyte: Research Funding; CTI BioPharma Corp: Research Funding; Promedior: Research Funding; Gilead: Research Funding; Celgene: Consultancy, Research Funding; NS Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; Sierra Oncology: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Roche: Research Funding.

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Integrative Analysis of FISH, Transcriptomics and Mutational Status Predicts Responsiveness to Novel Agents in Multiple Myeloma

Blood ◽

10.1182/blood-2019-130977 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 574-574

Author(s):

Cecilia Bonolo De Campos ◽

Caleb K Stein ◽

Nathalie Meurice ◽

Laura Ann Bruins ◽

Joachim L Petit ◽

...

Keyword(s):

Multiple Myeloma ◽

High Throughput ◽

Predictive Models ◽

Research Funding ◽

Ex Vivo ◽

Integrative Analysis ◽

Drug Screen ◽

Mrna Sequencing ◽

Bet Inhibitors ◽

Selection Of

Introduction Despite continuous improvement of clinical outcome in multiple myeloma (MM), disease relapse remains a major challenge, leading to progressively shorter remissions and fewer treatment options. Strategies attempting to counteract this challenge include recent efforts resulting in an increase in the availability of novel promising anti-MM agents and targeting specific genetic profiles of the disease. In this context, we aim to develop predictive models of sensitivity and resistance to novel compounds by connecting an ex vivo high-throughput drug screen with genetic, transcriptomics, FISH, and clinical features. Methods Twenty compounds (afatinib, afuresertib, belinostat, buparlisib, cobimetinib, CPI-0610, crenolanib, dinaciclib, dovitinib, JQ1, LGH447, osimertinib, OTX015, panobinostat, romidepsin, selinexor, sunitinib, trametinib, venetoclax, and vorinostat) were selected based on overall promising anti-MM activity from an ex vivo high throughput drug screen with a panel of 79 single agents incubated for 24 hours. The area under the curve (AUC) was used to rank order the ex vivo responses for each compound and the lowest and highest quartile samples were identified for further analysis. Clinical data and FISH data, including t(11;14), t(4;14), t(14;16), del(17p), +1q, monosomy 13, and MYC rearrangement, were collected. Targeted DNA sequencing was performed using a 2.3 Mb custom capture panel covering 139 MM-relevant genes. mRNA-sequencing was performed and differential gene expression analysis in the highest and lowest quartile identified subsets of markers positively and negatively associated with the AUC response for a given compound. An additional unbiased selection of markers using lasso techniques was performed, resulting in predictive generalized linear models (GLM) for each agent. Responses from the remaining intermediate samples were estimated with the predictive models, with overall predictive ability assessed by correlating predicted AUCs with their actual counterparts. Results Our integrative analysis was performed on 50 primary patient samples (36% untreated and 64% relapsed MM). Venetoclax, dinaciclib, romidepsin, panobinostat, osimertinib, belinostat and selinexor were the most active compounds in the cohort. Interestingly, LGH447, dovitinib, selinexor, JQ1, OTX-015, cobimetinib, and trametinib showed increased activity in relapsed MM when compared to untreated samples (Wilcoxon Test; p<0.05). We generated GLMs using an average of 92 markers (range 64-107) per compound, combining mRNA-sequencing expression with FISH and mutation data. The analysis proposed in the present study was validated through the unbiased selection of BCL2 among the subset of markers included in the GLM predicting sensitivity to venetoclax, a first-in-class orally bioavailable selective BCL2 inhibitor. Expression level of critical NF-kB and cell cycle genes, such as BIRC3, CKS1B, PAX5, NFKB2, and CCND2, were included in 60% of our predictive models. Mutations of DNA repair genes (ATM,TP53) were included in the GLMs of three epigenetic therapies, one histone deacetylase inhibitor and two BET inhibitors, associated to ex vivo resistance to the drugs. The presence of monosomy 13 was also a marker for ex vivo resistance for five epigenetic therapies, four HDAC inhibitors and one BET inhibitor. The three BET inhibitors, JQ1, CPI-0610, and OTX015, were among the compounds most accurately predicted by our integrative approach, with Spearman correlation values between 0.773-0.858. Overall, our models accurately predicted the ex vivo response for 16 (80%) of the compounds (r>0.7). Five (25%) of these compounds displayed a remarkably accurate prediction model in both training (highest and lowest quartiles) and validation (intermediate quartiles) samples (r>0.8). Conclusions The GLM data integration approach enabled the establishment of effective predictive models, identifying FISH, transcriptomics, and mutations of putative driver genes important in anti-MM agent responsiveness. In addition, the resulting dataset is promising for future research focusing on the discovery of novel mechanisms of action and establishing markers of sensitivity and resistance to novel compounds. We are currently increasing our dataset and seek to create an omnibus approach that predicts responses to multiple anti-MM agents simultaneously. Disclosures Bergsagel: Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Stewart:Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy; Ono: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Takeda: Consultancy.

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Targeted Exome Sequencing Identifies Novel Mutations in Familial Myeloproliferative Neoplasms Patients in the State of Qatar

Blood ◽

10.1182/blood.v124.21.5570.5570 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5570-5570 ◽

Cited By ~ 1

Author(s):

Nader I Al-Dewik ◽

Bruno Cassinat ◽

Jean-Jacques Kiladjian ◽

Alexander Knuth ◽

Mohamed A. Yassin

Keyword(s):

Exome Sequencing ◽

Research Funding ◽

Germ Line ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Genetic Alterations ◽

Molecular Techniques ◽

Research Fund ◽

Healthy Individuals ◽

Targeted Exome Sequencing

Abstract Background: Myeloproliferative Neoplasms (MPNs) are clonal hematopoietic disorders characterized by excessive proliferation of one or more myeloid cell lineages. Philadelphia negative MPNs include Polycythemia Vera (PV), Essential Thrombocytosis (ET) & Primary Myelofibrosis (PMF). MPNs are associated with the presence JAK2 V617F mutation in 95% of PV & 50% of ET & PMF patients. Several molecular techniques such as RQ-PCR, HRM & Sequencing are currently used to detect common mutations. However, there are still significant numbers of MPNs that are negative to the most common genetic anomalies & many mutations are still unknown. The advent of Next Generation Sequencing (NGS) gives the opportunity to study relevant mutations in several genes. Aim: Utilizing NGS to identify potential genetic anomalies causing familial MPNs patients in Qatar. Methods: 6 MPNs patients from consanguineous families & 5 healthy individuals were consented into the study & peripheral blood samples were collected. gDNA was extracted & used for multiplex PCR amplification of amplicons targeting cancer associated mutations in 28 key genes (JAK2, MPL, THPO, CBL, LNK, SH2B3, NF1, SOCS1/2/3, TP53, NRAS/KRAS, NF1, IDH1/2, EZH2, ASXL1, TET2, ATM, KIT, RB, TP53, IKZF1, RUNX1, PDGFRB, TERT & CALR) using the Ion AmpliSeq Kit. NGS was performed via the Ion Torrent using the 318 chip & data was analyzed with the Torrent Suite Software. Mutation details were obtained from COSMIC database. A hg 19 sequence was used as reference. The confirmation of NGS data was performed using RQ-PCR or Sequencing. Results: 11 samples were successfully sequenced, with a mean depth of 1500 reads & the FASTQC plugin indicated good quality sequencing metrics. JAK2 V617F, JAK2 exon 12-15 & MPL (S505N, W515 L/K) negative samples tested before via RQ-PCR, HRM & sequencing were called negative by NGS. NGS identified novel deleterious mutations in MPNs patients. Out of 6 familial cases, 5 patients (P1- P5) were ET & 1 patient (P6) was PV. P1 had JAK2 V617F, ASXL1 T600P, CBFB G180S, THPO S184R &ITGA2R76Q, P2 had JAK2 V617F, MPL A554G & ATM F582L, the other three Patients (P3, P4 & P5) had CLAR K385fs*47 & one PV patient (P6) had TYK2 E1163G, ASXL1 P808H, PDGFRB P4L & TERT G300fs. Among the patients & healthy individuals, mutations/SNVs such as MPL P106L, K553N, SH2B3 L476F, ATM F1036F KIT N564S & TET2 T730R were also found Discussion & conclusion: Initial screening of known common genes (JAK2 V617F, JAK2 exon 12-15 & MPL W515 L/K) mutations did not reveal the causative mutations in 3% of 180 PV patients, 52% of 200 ET patients & 77% of 20 PMF patients. In this study, several deleterious somatic & germ-line mutations & SNVs were identified using Targeted Exome Sequencing approach. A complex combination of mutations in JAK2, THPO, ITGA2 & MPL genes occurred in ET patients & coexistence of several oncogenic events in TYK2, ASXL1, PDGFRB & TERT occurred in PV patient. This ﬁnding may also suggest that the MPNs phenotype may depend on presence of other mutations. It is worth mentioning that the presence of ATM variant in P2 is associated with increased risk of CLL. Somatic CALR type-2 mutation was identified in 3 ET (nonmutated JAK2 or MPL) patients. This mutation is 5-bp TTGTC insertion in exon 9 that generates a mutant protein with a novel C-terminal (p.K385fs*47). In patients & healthy individuals, a heterozygous germ-line mutation in exon 3 of the MPL gene (MPL P106L) has been observed. it has previously been described as a rare autosomal-dominant disorder. However, this mutation is considered to be frequent in Arabic populations, leading to severe thrombocytosis in homozygotes & occasionally to mild thrombocytosis in heterozygotes. In addition, several unreported variants of uncertain significance were identified. Our preliminary results suggested that MPNs patients in Qatar have several potential disease- associated variants & mutations. Evidences show that there exists a possibility of the disease arising out of the accumulation of genetic alterations & not as the consequence of a single genetic-hit event. This could possibly be due to the high rate of consanguineous marriages in Qatar i.e. the "Founder Effect". Our results recommended carrying out WES to explore & identify mutations which will be crucial to characterize many cases of MPNs with unknown molecular causes, gain a deep understanding of genotype-phenotype correlations & MPNs pathogenesis. Disclosures Al-Dewik: Qatar National Research Fund: Patents & Royalties, Research Funding. Yassin:Qatar National research fund: Patents & Royalties, Research Funding.

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Rational cotargeting of HDAC6 and BET proteins yields synergistic antimyeloma activity

Blood Advances ◽

10.1182/bloodadvances.2018026484 ◽

2019 ◽

Vol 3 (8) ◽

pp. 1318-1329 ◽

Cited By ~ 7

Author(s):

Jennifer S. Carew ◽

Claudia M. Espitia ◽

Weiguo Zhao ◽

Valeria Visconte ◽

Faiz Anwer ◽

...

Keyword(s):

Ex Vivo ◽

Family Members ◽

Apoptosis Induction ◽

Histone Deacetylase 6 ◽

Novel Approach ◽

Bet Inhibitors ◽

Hdac6 Inhibitor ◽

Bet Protein

Abstract Inhibition of bromodomain and extra terminal (BET) protein family members, including BRD4, decreases the expression of c-MYC and other key oncogenic factors and also significantly induces histone deacetylase 6 (HDAC6) expression. On the basis of the role of HDAC6 in malignant pathogenesis, we hypothesized that rational cotargeting of HDAC6 and BET family proteins may represent a novel approach that yields synergistic antimyeloma activity. We used genetic and pharmacologic approaches to selectively impair HDAC6 and BET function and evaluated the consequential impact on myeloma pathogenesis. These studies identified HDAC6 upregulation as an efficacy reducing mechanism for BET inhibitors because antagonizing HDAC6 activity synergistically enhanced the activity of JQ1 in a panel of multiple myeloma (MM) cell lines and primary CD138+ cells obtained from patients with MM. The synergy of this therapeutic combination was linked to significant reductions in c-MYC expression and increases in apoptosis induction. Administration of the clinical HDAC6 inhibitor ricolinostat was very well tolerated and significantly augmented the in vivo antimyeloma activity of JQ1. Ex vivo pharmacodynamic analyses demonstrated that the combination of JQ1 and ricolinostat led to significantly lower MM cell proliferation and increased apoptosis and diminished expression of c-MYC and BCL-2. These data demonstrate that cotargeting of HDAC6 and BET family members is a novel and clinically actionable approach to augment the efficacy of both classes of agents that warrants further investigation.

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Mir-9 Is Aberrantly Expressed In MPN Patients and Accelerates Erythropoietic Cell Growth and Differentiation

Blood ◽

10.1182/blood.v116.21.1983.1983 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1983-1983

Author(s):

Xiaoqing Lin ◽

Kim Rice ◽

Martin Carroll ◽

Fabricio Costa ◽

Outi Kilpivaara ◽

...

Keyword(s):

Cell Growth ◽

Mirna Expression ◽

Research Funding ◽

Zinc Finger Protein ◽

Ex Vivo ◽

Myeloproliferative Neoplasms ◽

Erythroid Differentiation ◽

Gain Of Function ◽

Mrna Targets ◽

Cd34 Cells

Abstract Abstract 1983 The myeloproliferative neoplasms (MPNs), PV, ET and IMF, harbor a common gain-of-function mutation of JAK2V617F but in the past several years other molecular lesions have been noted as well such as mutation of TET2, chromsomal gains and losses, aberrant mRNA expression and aberrant microRNA expression. Since miRNAs are important regulators of hematopoiesis, we measured miRNA expression levels in CD34+ cells isolated from 8 patient samples (4 PV with JAK2V617F, 3 ET with wild-type JAK2 and 1 IMF with unknown JAK2 status) and 4 healthy controls using a Taqman Low Density Array (TLDA) representing 667 known miRNAs. We identified 28 miRNAs that were significantly deregulated in MPN patients compared to controls (p<0.05). Many of these miRNAs (18/24; 75%) were also deregulated in an independent miRNA array study using MPN patient granulocytes (n=39, 25 PV, 14 ET). Among these miRNAs, miR-9 expression was significantly upregulated in MPN patient CD34+ cells as well as in mature granulocytes. Furthermore, miR-9 expression was responsive to modulation of JAK2 activity in both gain of function (overexpression of JAK2V617F in TF-1 cells) and loss of function (inhibition of JAK2 activity in HEL cells) systems, suggesting that miR-9 may be dysregulated by aberrant JAK2V617F signaling. Analysis of miR-9 expression in ex-vivo, uni-lineage human CD34+ cultures, revealed that miR-9 is expressed at low levels during erythropoiesis, while levels gradually increase during myeloid differentiation. To determine how deregulated expression of miR-9 affected erythropoiesis, we transduced human bone marrow CD34+ cells (AllCells) with empty vector or miR-9 lentivirus, and examined erythroid differentiation and proliferation in liquid culture over 12 days. miR-9 expressing cells displayed a consistent growth advantage (∼30%), and accelerated erythroid differentiation (CD71+, GlyA+) at days 7 and 9. HITS-CLIP is a high throughput miRNA target prediction method developed in the Darnell lab based on crosslinking Argonaute protein to miRNA and target mRNA, providing highly specific identification of direct miRNA targets. We previously identified genes differentially expressed in PV patient CD34+ cells compared to normal CD34+ cells using Affymetrix HG-U133A arrays (117 genes, p<0.05). A comparison of predicted miR-9 mRNA targets identified using HITS-CLIP in P13 mouse brain, with genes downregulated in PV patient microarray studies, identified KLF4 which encodes a zinc finger protein involved in pluripotency and YWHAZ a 14-3-3 protein involved in signaling pathways regulating proliferation and survival as potential miR-9 targets relevant to aberrant hematopoiesis in MPN. Dysregulated cell growth in MPN may result in part from aberrant miRNA expression. Disclosures: Carroll: Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding.

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Modeling Calreticulin-Mutant Myeloproliferative Neoplasms with Isogenic Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood-2018-99-111512 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4319-4319 ◽

Cited By ~ 1

Author(s):

Wei Wang ◽

Tiansu Wang ◽

Andriana G. Kotini ◽

Camelia Iancu-Rubin ◽

Ronald Hoffman ◽

...

Keyword(s):

Research Funding ◽

Ex Vivo ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Mutant Type ◽

C Terminus ◽

Calr Mutations

Abstract Myeloproliferative neoplasms (MPN) are characterized by the excessive production of one or more myeloid lineages and a propensity to progress to acute leukemia. In 2013, mutations in the CALR gene, encoding calreticulin, were identified in patients with MPN, mutually exclusive to the previously identified JAK2 and MPL (TPO-R) mutations. CALR mutations are frameshift mutations - typically a 52-bp deletion (type 1) or a 5-bp insertion (type 2) - that result in a novel C-terminus. The discovery of mutations in a ubiquitously expressed multifunctional protein like calreticulin was unanticipated. Subsequent studies found that CALR mutations lead to activation of JAK/STAT, mediated through aberrant interactions between mutant CALR and MPL, thus presenting an excellent opportunity for targeted therapy. However, the mechanism of MPL activation remains largely unexplained with prior studies using cell lines with exogenous expression of CALR and MPL following transfection. To create a more physiological cellular model to study the effects of CALR mutations, we established multiple iPSC lines from two patients with CALR-mutant MPN - one type 1-like (del34) and one type 2 (ins5) -, as well as from one patient with JAK2V617F MPN. All iPSC lines were confirmed to harbour the CALR or JAK2V617F mutation found in the corresponding patient, to express mutant calreticulin, as detected by flow cytometry using an antibody which specifically recognizes the novel calreticulin C-terminus, and to be karyotypically normal. Genetically matched iPSC lines with WT JAK2 could also be generated from the JAK2V617F (but not the CALR-mutant) patient cells in the same reprogramming round. CRISPR gene editing was used to generate isogenic CALR-corrected lines from both CALR-mutant patients. Furthermore, in order to facilitate biochemical studies, we used CRISPR to introduce a V5 epitope tag in one allele of the endogenous mutant or WT CALR gene, in mutant and isogenic corrected iPSC lines, respectively. We optimized an in vitro differentiation protocol for efficient derivation of megakaryocyte (MK) progenitors from iPSCs and found disease-relevant phenotypes, mainly TPO-independent MK colony formation in semi-solid media, which is the phenotypic hallmark of ex vivo primary MPN cells. In the absence of TPO, JAK2 V617F, CALR-mutant type 1-like and CALR-mutant type 2 iPSCs generated 52.1%, 58.7±22.2% and 59.8±3.6%, respectively, of the number of MK colonies generated in the presence of TPO, as opposed to 10%, 8.8±1.8% and 0.5±0.9%, respectively, for the matched WT JAK2, the corrected CALR-mutant type 1-like and the corrected CALR-mutant type 2 iPSCs. Isolated CALR mutant iPSC-derived CD41a+ MK progenitors had increased phosphorylation of STAT5 following cytokine starvation as compared to isogenic corrected and non-isogenic normal cells. CALR-mutant cells expressed equal transcript levels of the WT and mutant CALR alleles. However, mutant CALR protein levels were severely reduced, at levels 1~12% of those of the WT protein. This is consistent with previous studies documenting instability of mutant calreticulin. Transcriptomics (RNA-seq) and proteomics analyses of CD41a+-sorted MK progenitors derived from CALR mutant and isogenic corrected iPSCs are ongoing. These iPSC models offer the opportunity to study the effects of CALR mutations in a cellular context with both MPL and CALR (WT or mutant) expressed from their endogenous loci. They thus provide a powerful platform to investigate the disease mechanisms underlying CALR-mutant MPNs and to perform small molecule and genetic (CRISPR) screens to identify new therapeutic targets. Disclosures Iancu-Rubin: Merck: Research Funding; Incyte: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Merus: Research Funding; Formation Biologics: Research Funding; Janssen: Research Funding; Summer Road: Research Funding.

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Use of Pegylated Interferon in Six Pediatric Patients with Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2019-124023 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4194-4194 ◽

Cited By ~ 1

Author(s):

Nicole Kucine ◽

Shayla Bergmann ◽

Spencer Krichevsky ◽

Devin Jones ◽

Michael E. Rytting ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Pediatric Patients ◽

Myeloproliferative Neoplasms ◽

Adult Patients ◽

Specific Information ◽

Advisory Committees ◽

Oncology Research ◽

Increased Risk ◽

Future Fertility

Introduction: The classical BCR-ABL1 negative myeloproliferative neoplasms (MPNs) are clonal disorders of marrow overproliferation. Clinical phenotypes include polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). In adults, both risk stratifications and treatment guidelines are available to physicians. Various agents, including clinical trial agents, are utilized for treatment in adults. Conversely, there are no management guidelines for children with MPNs, and therapeutic options are limited. Hydroxyurea (HU) is the most commonly used agent in children, likely because of physician experience using this medication for sickle cell anemia. Children with MPN have been treated with HU with effective cytoreduction, although its use is controversial because of the concern for leukemogenicity. While it is still considered as first-line therapy in adults with MPNs, some guidelines recommend avoiding hydroxyurea in younger adult patients. Interferon (IFN) has been utilized in adults with MPNs for many years, and pegylated forms (PEG) have made the medication more tolerable. Enthusiasm in IFN is increasing, due to reports in adult patients of hematologic remissions and significant molecular responses, including molecular remissions in rare cases. Given its improved tolerability and potential benefits, IFN may be an appropriate choice for treatment in many children with MPNs. Given the limited experience in this population and available literature, we sought to review a small cohort of pediatric MPNs treated with PEG. Methods: We reviewed the charts of pediatric patients with MPNs who have been treated with PEG and pooled de-identified data. Demographic, mutational, laboratory, and treatment-specific information was documented. This study was approved by institutional IRBs. Results: Six children were identified at four institutions who had been treated with IFN (Table 1). Age at diagnosis of MPN ranged from 2-14 years, and age at onset of PEG therapy ranged from 3-16 years. Two children are female, and four are male. Three children were diagnosed with PV and three with ET. One child with PV is positive for a JAK2 Exon 12 mutation, three children have JAK2V617F mutations (two with PV and one with ET), one child with ET has a CALR mutation, and one child has triple-negative ET. Cytoreductive therapy was started in this cohort for a variety of reasons, including worsening counts, concerning marrow findings, and symptoms (headache being the most common.) Three of the children in this cohort had been treated with HU prior to starting PEG. Reasons for switching cytoreduction to PEG included family concern for increased risk of malignancy and concern for negative effects on future fertility, interest in a disease-modifying agent, and poor side-effect profile of HU. PEG doses ranged from 45 to 90 mcg per dose (Table 2), with dosing frequency ranging from one dose every 1-4 weeks. Blood counts remained generally stable or improved in this cohort; no children required stopping and switching to another cytoreductive agent. One subject developed a pulmonary embolism while on PEG, but was able to remain on therapy; no other MPN-related complications occurred while on PEG. Some children experienced improvement in clinical disease-related symptoms. Mild side-effects reported by subjects included headache, flu-like symptoms, injection site reaction, and abdominal pain. One child had PEG dosed less frequently because of issues with depression and anxiety, and ultimately had therapy discontinued due to normalization of platelet counts. No other dose-limiting drug-related toxicity was reported. All other subjects remain on therapy with PEG, and the duration of therapy ranges from 3-168 months. Conclusion: This cohort of young MPN patients has been treated with PEG with no major dose-limiting toxicity, and with sustained tolerability. While further study is needed, it is clear that PEG can have a role to play in the management of children with MPNs. Having multiple options for cytoreduction available for families to discuss with their pediatric practitioners allows for greater family autonomy. Our results to date highlight the need for prospective study of a larger group of young patients with MPNs treated with PEG. Disclosures Bergmann: Novartis: Research Funding, Speakers Bureau; Genentech: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Shire/Takeda: Research Funding; NovoNordisk: Research Funding. Mascarenhas:Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merus: Research Funding; Promedior: Research Funding; Janssen: Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche: Consultancy, Research Funding; Merck: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Verstovsek:Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy; Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding. Hoffman:Merus: Research Funding. OffLabel Disclosure: Use of interferon and hydroxyurea in children with MPNs is off-label

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Genomic Landscape of Young ATLL Patients Identifies Frequent Targetable CD28 Fusions

Blood ◽

10.1182/blood-2019-122525 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2760-2760

Author(s):

Noriaki Yoshida ◽

Kay Shigemori ◽

Samuel Y. Ng ◽

Ajit Nirmal ◽

Nicolas Donaldson ◽

...

Keyword(s):

T Cell ◽

Research Funding ◽

Young Patients ◽

Genetic Alterations ◽

Gene Set Enrichment Analysis ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Cell Leukemia Virus Type ◽

Raji Cells ◽

Oncology Research

Introduction Adult T-cell leukemia/lymphoma (ATLL) is a T-cell neoplasm induced by human T-cell leukemia virus type 1 (HTLV-1). In endemic areas, HTLV-1 infection typically occurs during breastfeeding but the median age of ATLL presentation in Japan is >70. Approximately 5% of ATLL patients in Japan present at age <50. Here, we hypothesized that ATLL in younger patients will have distinct genetic alterations. Methods DNA and RNA samples were extracted from frozen lymph nodes. Targeted capture sequencing for 98 genes and analysis of RNA sequencing (RNAseq) were performed in 8 samples of young patients with ATL (defined as ≤ 50 years in the current study) Results and discussion The most frequently mutated genes were CCR4 and CARD11, each in 3 of the analyzed cases (37.5%). Mutations of PLCG1, PRKCB and STAT3 were frequent in a previous genomic analysis (Kataoka et al. Nat Genet. 2015) that were not age-selected, but were not identified in our 8 cases. Three (37.5%) cases harbored concurrent CTLA4-CD28 and ICOS-CD28 fusions. This contrasts with previous reports, in which cases with both fusions were found in <1% of PTCL and ATLL patients. We confirmed the presence of both fusions in all 3 cases by RT-PCR. The structure of the CTLA4-CD28 fusion suggests that the extracellular portion of CTLA4 is expressed on the cell surface where it can interact with CD80 and CD86 to activate signaling through the intracellular CD28 portion. In contrast to CTLA4-CD28, the ICOS-CD28 fusion links the N-terminal signal peptide of ICOS with the extracellular and intracellular portions of CD28. This fusion should simply result in overexpression of CD28 and haploinsufficiency of ICOS. To evaluate the function of CTLA4-CD28, we used Ba/F3 cells, which are strictly dependent on exogenous IL-3 for proliferation. We transduced Ba/F3 cells with CTLA4-CD28 or a CTLA4-CD28 mutant (mut) with three amino acid substitutions that abrogate CD28 signaling. We then cultured these cells in the presence or absence of Raji cells, which express CD80 and CD86. In the absence of Raji cells, neither CTLA4-CD28 nor CTLA4-CD28mut expression conferred IL-3-independent growth. However, Ba/F3 cells expressing CTLA4-CD28 cultured in the presence of Raji cells achieved IL-3 independence. This was not the case for Ba/F3 cells expressing CTLA4-CD28mut, indicating a requirement on functional CTLA4-CD28 signaling. Western-blot analysis identified activation of CD28 signal in Ba/F3 cells with CTLA4-CD28 co-cultured with Raji cells. These data indicate that ligand-bound CTLA4-CD28 can promote downstream signaling in the absence of an endogenous TCR complex. Gene set enrichment analysis (GSEA) comparing the 3 ATLL cases with CD28 fusions to the 5 cases that lacked fusions demonstrated enrichment of previously defined gene signatures associated with AKT and RAF signaling, both of which are downstream of CD28 activation. GSEA also identified enrichments of T-cell function-related signatures, including pathways involved in interferon responses, in cases with CD28 fusions Cases with CTLA4-CD28 and ICOS-CD28 fusions become haploinsufficient for CTLA4 and ICOS. As expected, these cases had lower expression of both genes by RNAseq. Interestingly, these cases also had higher expression of CD80. By immunohistochemistry, ATLL cells with CTLA4-CD28 and ICOS-CD28 fusions expressed CD80 and macrophages in the tumor microenvironment expressed CD86. Thus, both intra- and intercellular interactions could drive CTLA4-CD28 and ICOS-CD28 signaling in these cases. Treatment of Ba/F3 cells expressing CTLA4-CD28 and cultured in the presence of Raji cells with a CTLA4 blocking antibody suppressed proliferation in a dose-dependent fashion. A previous case report (Mol Genet Genomic Med, 2015) described a patient with Sezary Syndrome and CTLA4-CD28 fusion who had a deep but transient response to the anti-CTLA4 antibody ipilimumab. This study did not clarify the extent to which response resulted from blocking of cell-autonomous CTLA4-CD28 signaling versus activation of a cell non-autonomous immune response. Nonetheless, it strongly supports the testing of CTLA4 blockade in additional cases of T-cell malignancies, including ATLL, that harbor CTLA4-CD28 fusions. Disclosures Stevenson: Celgene: Research Funding. Ohshima:Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; NEC Corp.: Research Funding; SRL, Inc.: Consultancy. Weinstock:Verastem Oncology: Research Funding; Celgene: Research Funding.

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Reversibility of the Resistance to Lenalidomide and Pomalidomide and Absence of Cross-Resistance in a Murine Model of MM

Blood ◽

10.1182/blood.v118.21.134.134 ◽

2011 ◽

Vol 118 (21) ◽

pp. 134-134 ◽

Cited By ~ 1

Author(s):

Enrique M Ocio ◽

Diego Fernández-Lázaro ◽

Laura San-Segundo ◽

Lorena González-Méndez ◽

Montserrat Martín-Sánchez ◽

...

Keyword(s):

Research Funding ◽

Tumor Volume ◽

Ex Vivo ◽

Acquired Resistance ◽

Resistance Mechanisms ◽

Continuous Treatment ◽

Cross Resistance ◽

Initial Growth ◽

To Receive

Abstract Abstract 134 Introduction: The introduction of novel agents has improved the outcome of MM patients, but MM is still considered an incurable disease and the emergence of resistance is the main responsible for this situation. The unraveling of the resistance mechanisms would help to design novel therapeutic strategies (combinations or sequencing treatments) to overcome this problem. Material and Methods: We have developed an in vivo model of acquired resistance to antimyeloma agents based in a model of subcutaneous plasmocytoma (MM1S) in CB17-SCID mice. For this purpose, mice were treated with Lenalidomide (25 mg/Kg) + Dex (1 mg/Kg) (LD), Pomalidomide (5 mg/Kg) + Dex (PD) or vehicle control, and after a period of initial sensitivity, tumors developed resistance to the administered combination. At this moment selected sensitive and resistant tumors were excised to analyze apoptosis, signaling pathways and gene expression profiling (GEP) changes. Moreover, some of these mice bearing resistant tumors were switched to receive the alternative combination (that is LD-PD or PD- LD), and, in selected mice, a second change of treatment was performed after secondary resistance to receive again the initial treatment (LD-PD-LD or PD-LD-PD). In order to evaluate TTP and to define the moment to change treatment we considered progression when tumor volume reached 1.700 mm3. Results: Both combinations (LD & PD) controlled the initial growth of the tumors, with a higher potency for the PD combination. Tumor volume reached 500 mm3 at a median of 8, 42 & 53 days for control, LD and PD respectively (p=0.01 and p=0.001). Nevertheless, after 30 days of continuous treatment, and despite maintaining the administration of the drugs, tumors started growing, and, once the tumors had reached 500 mm3, their growth kinetic was similar for the treated mice (despite still being treated) as compared to the untreated mice, indicating the emergence of complete resistance. This resistance was also confirmed ex vivo by in vitro culture with the corresponding drugs. In order to test the presence of cross-resistance, mice bearing big tumors resistant to LD or PD and already growing in an exponential phase (volume of 1.700 mm3), were at this point treated with the alternative combination. This sequential treatment change induced tumor stabilization and even a decrease of tumor volume. Again PD was significantly more potent at overcoming LD resistance as compared to the alternative situation, and this was verified both in terms of tumor growth inhibition (p=0.005) and in terms of time to progression (median of 16 vs 27 days for LD and PD respectively. p=0.004). Furthermore, mice that had been treated with LD-PD or PD-LD and had developed resistance to both combinations were again treated with the initial combination, and, surprisingly, they were again sensitive, indicating the reversibility of the acquired resistance. Similarly to previous experiments, PD was again significantly more potent than LD. This reversibility was also confirmed ex vivo after culture of the cells in medium without drugs. In order to investigate the resistance mechanisms, cells extracted from sensitive and resistant tumors were analyzed by Western Blot. Treatment with LD and PD induced a downregulation of pERK 1/2, nevertheless, when these cells developed resistance a very significant increase of pERK 1/2 levels (even higher than the basal levels) was observed. Moreover, these resistant cells also showed an upregulation of p-MEK, p-RAF and RAS. In this same line, the MEK inhibitor PD-98059 potentiated the in vitro activity of LD or PD in fresh MM1S cells, with high synergism (CI<0.1). Finally, changes in GEP were evaluated in extracted tumors. A significant change was observed in cells from tumors sensitive to PD and LD (with a higher gene deregulation with the former combination), as compared to untreated ones. Interestingly, when tumors became resistant, most of these changes disappeared and the GEP partially returned to a profile similar to that of active untreated tumors. Conclusion: The data presented would support treatment with alternative IMIDs if resistance was developed to one of them (specially PD was significantly more potent at rescuing resistance to LD), or even the retreatment with the same IMID after a wash up period. Moreover this study supports the evaluation of combinations of IMIDs with agents that abrogate the ERK pathway in order to increase efficacy or avoid resistance. Disclosures: Quintana: Celgene: Employment. García:Celgene: Employment. Pandiella:Celgene: Research Funding. San Miguel:Celgene: Consultancy, Research Funding.

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