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

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5570-5570 ◽  
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.

High Percentage of JAK2 exon 12 Mutation in Polycythemia Vera and Essential Thrombocythemia Among Populations of Qatar

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5064-5064
Author(s):  
Mohamed A. Yassin ◽  
Hanadi Rafii El-Ayoubi ◽  
Nader Al-Dewik
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Genomic Dna ◽  
Research Funding ◽  
Mutant Allele ◽  
Jak2 V617f ◽  
Research Fund ◽  
Idiopathic Myelofibrosis ◽  
Essential Thrombocytosis ◽  
High Incidence

Abstract Abstract 5064 The chronic myeloproliferative Neoplasm (NPM) are clonal hematopoietic stem cell malignancies with 3 main subtypes: polycythemia vera (PV), essential thrombocytosis, and idiopathic myelofibrosis. PV is characterized by increased RBC proliferation in the absence of erythropoietin and proliferation of myeloid lineages usually is noted, A gain-of-function mutation of Janus kinase 2 (JAK2) V617F, is identified in about 95% of patients with PV and about 50% of patients with essential thrombocytosis and idiopathic myelofibrosis. It has been shown that JAK2 exon 12 mutations can activate erythropoietin signaling pathways while these findings have been confirmed by many studies from Western countries, there are no reports from Asian countries in general and Arab countries in particular about the prevalence of the JAK2 exon 12 mutation in patients with PV and ET. In the present study, we determined the prevalence of JAK2V617F and JAK2 exon 12 mutations in patients with PV and ET in Qatar. Materials and Methods We enrolled patients with a diagnosis of PV and ET at National Centre for Cancer Care and Research in Qatar from January till June 2012. The diagnosis of PV and ET was established according to the 2008 World Health Organization criteria. The study included 82 patients. Clinical information and the CBC data at diagnosis were obtained from medical records. Pretreatment serum erythropoietin levels. Total DNA was isolated from buffy coat cells taken from peripheral blood using a kit (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer's instructions. Allele-specific polymerase chain reaction (PCR) was performed using 80 ng of genomic DNA as the template in a35-cycle PCR reaction at an annealing temperature of 58°C, as previously described. The mutant allele yields a 203-base-pair (bp) PCR product (sensitivity of mutant allele detection <1%). For exon 12 mutation screening, 80 ng of genomic DNA was amplified by specific primers designed to amplify a region of 453 bp containing the 128 bp of the exon 12 sequence of JAK2. PCR products were directly sequenced in both directions on an ABI 3730 DNA Analyzer using the BigDye Terminator Sequencing kit. Results We examined the occurrence of JAK2V617F and JAK2 exon 12 mutations in a clinical cohort of 82 patients with polycythemia vera (PV) and Essential thrombocythemia (ET) Of which 42 patients had PV aged 25 to 53, 13 (31%) females and 29 (69 %) males and V617F mutation was detected in all of them exon 12 mutation was detected in 38 (90. 47%) patients. We found 2 different exon 12 mutations:3 N542-E543del, 1 F537-K539delinsL, and among 40 ET patients aged 25 to 59, 22 (55 %) males and 18 (45%) females, 35 patients (87. 5%) were JAK2 V617F and JAK 2 exon12 positive and 5 (12. 5%) were JAK2V617F as well as exon 12 negative patients with V617F and exon 12 mutations showed significantly higher WBC and platelet counts at diagnosis than patients with exon V617F mutation alone (P =. 021 and P =. 038, respectively). We report a surprisingly high incidence of exon 12 mutations in MPN patients with PVand ET in Qatar, a result quite different from reports in the Western literature (P =. 001). Conclusion Our data suggest that exon 12 mutation of JAK2 in patients with PV and ET may have an uneven geographic distribution. A clinical laboratory providing the V617F test alone may risk missing a substantial number of patients with PV in areas with a high incidence of exon 12 mutation. the importance of such associations may need further studies and evaluations. Disclosures: Yassin: Qatar National Research Fund: Patents & Royalties, Research Funding. Rafii El-Ayoubi:Qatar National Research Fund: Patents & Royalties, Research Funding. Al-Dewik:Qatar National Research Fund: Patents & Royalties, Research Funding.

scholarly journals BCR-ABL1 p190 in CML: A Minor Breakpoint with a Major Impact

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 190-190
Author(s):  
Shady Adnan Awad ◽  
Helena Hohtari ◽  
Komal Kumar Javarappa ◽  
Tania Brandstoetter ◽  
Daehong Kim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Exome Sequencing ◽  
Rna Sequencing ◽  
Cell Lines ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Genetic Alterations ◽  
Resistance Testing ◽  
Sequencing Analysis ◽  
Distinct Features

Introduction: The oncoprotein Bcr-Abl has two major isoforms, depending on the breakpoint in BCR gene, p190 and p210. While p210 is the hallmark of chronic myeloid leukemia (CML), p190 occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients. p190 occurs as a sole transcript in 1-2% CML patients, associated with distinct features like monocytosis and frequent additional cytogenetic abnormalities (ACA) at diagnosis. It also confers a risk of treatment failure and progression in chronic phase (CP) CML patients. However, the underlying mechanisms are largely unknown. Here we explore the characteristics of p190 and p210 in CML and ALL patients using next generation sequencing, phospho-flowcytometry and high throughput drug testing. Patients and methods: Peripheral blood mononuclear cells (PMNC) were collected at diagnosis from four CP-CML patients harboring p190 isoform from Helsinki University Hospital. Genetic alterations were identified by whole exome sequencing. RNA sequencing was employed to analyze transcriptional profiles of p190 CML (n=3) in contrast to p210 CML patients (n=4). A thorough transcriptional, phosphorylation and drug sensitivity profiling were applied to five p190- and three p210-expressing Ph+ALL patients. Expression alterations were further characterized in two cell line models mimicking BCR-ABL positive leukemia (Ba/F3 and HPCLSK). Phosphorylation profiles were analyzed by flowcytometry and phospho-array (Tyrosine Phosphorylation ProArray, Full Moon Biosystems). For drug sensitivity and resistance testing (DSRT), a custom plate set comprising 75 approved and investigational oncology drugs was used for patient samples and more extensive 528-drugs plates were used for the cell lines. Results: CML patients with p190 had a median age of 72.5 years at the diagnosis (range: 50-80) and all received imatinib as a frontline treatment. Only one patient achieved a fluctuating major molecular response (MMR) by 12 months while the rest of the patients showed primary resistance to treatment and were shifted to a 2nd line TKI, nilotinib (n=2) or proceeded to HSCT (n=1). By exome sequencing we identified 26 variants in p190-CML samples (median per patient=7, range: 2-10), including variants in ASXL1, DNMT3A and KDM4D genes. RNA-sequencing analysis identified 19 and 97 dysregulated genes (Q &lt;0.05) between p190- and p210 in CML and Ph+ ALL cells respectively. In CML, enrichment analysis revealed upregulation of TNF, interferon (IFN), IL1-R and Toll-like receptor (TLR) signaling, TP53-related, cell cycle and apoptosis pathways. Among Ph+ ALL samples, many CML-related genes were upregulated in samples encompassing p210 while IFN-, TP53- and cell cycle-related molecules were upregulated in p190 samples. p190 samples exhibited hyper-phosphorylation of Src kinase compared to p210 samples. DSRT results also revealed increased sensitivity of primary Ph+ ALL-p190 cells to Src-inhibitors (dasatinib and saracatinib), glucocorticoids and MDM2 inhibitors/TP53 activators (SAR405838 and idasanutlin). Regarding cell lines, Ba/F3-p190 showed the upregulation of interferon signaling pathways compared to p210. Src was also hyperphosphorylated in both Ba/F3 and HPCLSK p190 models. In addition to glucocorticoids and Src-inhibitors, compounds blocking the activity of the inhibitors of apoptosis protein (IAP) family were highly effective at reducing the viability of p190 compared to p210 cells in both cell lines. Conclusions: In CML, p190 isoform of BCR-ABL1 is associated with distinct features and should be considered as a high-risk group. Combining clinical, genomic, phosphorylation and drug sensitivity data, we demonstrated that p190 activates specific cancer pathways, notably Src signaling and interferon pathways. Data also suggests that CML patients with p190 could benefit from broad spectrum TKI with Src inhibiting activity or combination of TKI with MDM2- or IAP-inhibitors. Disclosures Heckman: Orion Pharma: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding. Porkka:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding. Mustjoki:Novartis: Research Funding; Pfizer: Research Funding; BMS: Honoraria, Research Funding.

scholarly journals RMC-4550, an Allosteric Inhibitor of SHP2, Displays Therapeutic Efficacy in Pre-Clinical Models of Myeloproliferative Neoplasms

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4198-4198 ◽  
Author(s):  
Garima Pandey ◽  
Nathan Horvat ◽  
Narmin E. Amin ◽  
Afua A. Akuffo ◽  
Christelle Colin ◽  
...  
Keyword(s):  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Critical Role ◽  
Philadelphia Chromosome ◽  
Jak2 V617f ◽  
Ras Signaling ◽  
Jak2 Inhibitor ◽  
Stat Signaling ◽  
Drug Naïve

Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) are JAK2-driven disorders resulting from mutations in JAK2, MPL, or CALR. Ruxolitinib, the only FDA-approved JAK2 inhibitor for MPNs, alleviates patient symptomology and improves quality of life, but has little effect on reducing mutant allele burden. This persistent survival of MPN cells in the face of ruxolitinib, as well as other JAK2 inhibitors that have been clinically tested, is a major clinical bottleneck to the development of an effective targeted therapy for MPN patients. Identifying new therapeutic targets which play critical roles in MPN cells and/or in JAK2 inhibitor persistence may lead to improved MPN therapies. SHP2 is an oncogenic tyrosine phosphatase that is an effector of growth factor and cytokine receptor signaling. SHP2 plays a critical role in the activation of the RAS-ERK pathway and regulates JAK-STAT signaling via numerous phosphatase-dependent mechanisms. Activating mutations of SHP2(PTPN11) have been identified in leukemia, including 8% of MPN patients whose disease progressed to acute myeloid leukemia (AML). In addition, SHP2 has been shown to mediate adaptive resistance to targeted therapies in several cancers. Given the role of SHP2 in cytokine and JAK-STAT signaling, we envisaged a potential role of SHP2 in MPN cell growth and/or survival and ruxolitinib persistence. Treatment of JAK2-V617F-driven MPN model cell lines (UKE1, SET2, and BaF3-JAK2-V617F) with ruxolitinib blocked constitutive tyrosine phosphorylation of SHP2, including phosphorylation of Y542, a marker for activated SHP2. This phosphorylation, however, was restored in ruxolitinib persistent cells. Combination treatment of the allosteric SHP2 inhibitor RMC-4550 (Revolution Medicines) with ruxolitinib prevented the development of ruxolitinib persistent cells and pre-established persistent cells remained sensitive to SHP2 inhibition. RMC-4550 treatment led to significantly reduced levels of pERK consistent with the role of SHP2 in RAS signaling. Interestingly, pERK levels in persistent cells were more sensitive to SHP2 inhibition compared to drug naïve cells suggesting pERK was more dependent on SHP2 in ruxolitinib persistent cells. SHP2 inhibitor treatment increased pSTAT5(Y694) in drug naïve cells but this increase was not observed in similarly treated persistent cells. Furthermore, while ruxolitinib inhibited pERK levels in UKE1 and SET2 cells, pERK levels recovered within 24 hrs of treatment. SHP2 inhibition prevented the recovery of pERK in the presence of ruxolitinib. Collectively, these data suggest that signaling pathways in MPN cells treated with ruxolitinib can become rewired, gaining greater dependence on SHP2, concomitant with sustained pERK and cell survival/growth. Interestingly, we identified a known activating SHP2 mutation (F71L) in UKE1 cells obtained from two independent sources - consistent with the presence of PTPN11 mutations in post-MPN AML. The persistent survival of UKE1 cells in ruxolitinib was antagonized by CRISPR-mediated reduction of SHP2 expression, providing further evidence that SHP2 contributes to ruxolitinib persistence. To assess the effects of a SHP2 inhibitor on MPN progression in vivo, we employed the MPLW515Lbone marrow transplant mouse model of MPN. Initial assessment of therapeutic treatment of mice with an established MPN phenotype indicated that once daily treatment of RMC-4550 (10 or 30 mg/kg) antagonized the MPN phenotype. Complete blood counts indicated a significant reduction in white blood cells, monocytes, and neutrophils compared to vehicle treated mice, while flow cytometry analysis indicated RMC-4550 diminished CD11b+ cell numbers to near that observed in mice transplanted with MPLWT-transduced bone marrow. RMC-4550 improved the overall health of diseased mice, as indicated by increased weight, and significantly reduced organomegaly of the spleen and liver compared to vehicle treated MPN mice. Finally, erythropoietin independent erythroid colony formation of JAK2V617F-positive MPN patient cells was suppressed following SHP2 inhibition, which synergized or enhanced the inhibition induced by ruxolitinib in this assay. In summary, our results suggest that SHP2 inhibition may represent a potential MPN therapy in both ruxolitinib naïve and resistant patients and is an attractive therapeutic target for future clinical investigation. Disclosures Epling-Burnette: Incyte Corporation: Research Funding; Forma Therapeutics: Research Funding; Celgene Corporation: Patents & Royalties, Research Funding. Reuther:Incyte Corporation: Research Funding.

scholarly journals Evaluation of genetic alterations in biliary tract cancer using targeted exome sequencing

2018 ◽  
Vol 29 ◽  
pp. viii257-viii258
Author(s):  
H. Chae ◽  
C. Yoo ◽  
D. Kim ◽  
J.H. Jung ◽  
H.-M. Chang ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Biliary Tract ◽  
Biliary Tract Cancer ◽  
Genetic Alterations ◽  
Targeted Exome Sequencing ◽  
Tract Cancer

scholarly journals Targeted exome sequencing profiles genetic alterations in leiomyosarcoma

2015 ◽  
Vol 55 (2) ◽  
pp. 124-130 ◽  
Author(s):  
Narasimhan P. Agaram ◽  
Lei Zhang ◽  
Francois LeLoarer ◽  
Tarik Silk ◽  
Yun-Shao Sung ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Genetic Alterations ◽  
Targeted Exome Sequencing

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

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4319-4319 ◽  
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.

scholarly journals Genetic Resistance to JAK2 Enzymatic Inhibitors Is Overcome by HSP90 Inhibition

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 62-62
Author(s):  
Oliver Weigert ◽  
Andrew A. Lane ◽  
Liat Bird ◽  
Nadja Kopp ◽  
Angela V Toms ◽  
...  
Keyword(s):  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Lymphoblastic Leukemia ◽  
Jak2 V617f ◽  
Kinase Domain ◽  
Janus Kinase ◽  
Jak Inhibitor ◽  
Resistance Mutations ◽  
Hsp90 Inhibitors ◽  
Imatinib Resistance

Abstract Abstract 62 Mutation within the kinase domain of tyrosine kinases is a common mechanisms of resistance to enzymatic inhibitors. Inhibitors of janus kinase 2 (JAK2) are under evaluation in patients with myeloproliferative neoplasms (MPNs), B-cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit CRLF2, and other tumors with constitutive JAK2 signaling. To identify resistance mutations in JAK2, we randomly mutagenized human JAK2 R683G, which is observed in approximately half of CRLF2-rearranged B-ALL. We transduced the mutagenized JAK2 cDNA library into murine Ba/F3 cells that express CRLF2. Expression of CRLF2 and JAK2 R683G confers IL3 independent growth in Ba/F3 cells. The transduced population was selected in the JAK2-selective inhibitor NVP-BVB808 in the absence of IL3. Multiple BVB808-resistant clones were recovered that harbored either E864K, Y931C or G935R mutations in JAK2. Alignment of homologous regions of the JAK2 kinase domain (JH1) with ABL1 demonstrated that the three mutations are located in regions homologous to imatinib resistance hotspots in ABL1. Codons Y931 and G935 are within the hinge region of the kinase domain. Based on structural modeling, Y931C is likely to inhibit substrate binding. E864K is located in the middle of b3 following the P-loop in the N-lobe and may modify the structure and flexibility of the preceding P-loop, thus destabilizing the conformation required for inhibitor binding. We expressed JAK2 V617F alleles harboring Y931C, G935R or E864K in Ba/F3-EPOR cells and exposed the cells to the JAK2 enzymatic inhibitors JAK inhibitor-1, NVP-BSK805, TG101348, tofacitinib (formerly tasocitnib), ruxolitinib (formerly INCB18424) and BVB808. All three mutations conferred 2- to >10-fold resistance against BVB808, NVP-BSK805, TG101348, ruxolitinib and JAK inhibitor-1. Y931C and E864K but not G935R conferred resistance to tofactinib. Modeling of G935R indicated that a 935R side-chain would occlude the hydrophobic channel of the ATP-binding pocket. As a consequence, this mutation would decrease the binding affinity of compounds occupying the hydrophobic channel like JAK inhibitor-1 or BSK805, but not affect the potency of tofactinib, which does not bind in this region. Mutation of G935 to arginine, histidine or glutamine reduced the inhibitory effects of JAK inhibitor-1, but not tofacitinib, on JAK2 kinase domain activity. None of the codon 935 mutations had significant effects on Km or Vmaxin vitro. BVB808 treatment partially reduced activation state-specific phosphorylation of STAT5 in Ba/F3-EPOR/JAK2 V617F cells but not in Ba/F3-EPOR/JAK2 V617F/G935R or G935H cells. JAK2 is a known client of HSP90, and HSP90 inhibitors promote the degradation of both wild-type and mutant JAK2. We hypothesized that resistance mutations within the JAK2 kinase domain would not affect JAK2 degradation induced by HSP90 inhibitors. We assayed the cytotoxicity of the resorcinylic isoxazole amide NVP-AUY922 and the benzoquinone ansamycin 17-AAG in Ba/F3 cells that express the erythropoietin receptor (EPOR) and JAK2 V617F, which is observed in more than half of MPNs. Mutation of JAK2 V617F to include E864K, Y931C or G935R did not affect sensitivity to either AUY922 or 17-AAG. In fact, AUY922 was more active against cells harboring G935R (GI50, 3.87 nM) or E864K (GI50, 6.14 nM) compared to cells with no resistance mutation (GI50, 14.7 nM; p<0.05). Both HSP90 inhibitors had similar potency in Ba/F3-CRLF2 cells expressing the resistance mutations in cis with R683G. Treatment of both lines with AUY922 at levels achievable in vivo reduced P-JAK2, P-STAT5, and total JAK2 regardless of mutations that conferred resistance to enzymatic JAK2 inhibitors. Thus, HSP90 inhibitors maintain activity in JAK2-dependent cells with resistance mutations in JAK2. Treatment of JAK2-dependent cancers with HSP90 inhibitors is an attractive treatment strategy either up-front or upon the selection of resistance to JAK2 enzymatic inhibitors. Disclosures: Gaul: Novartis: Employment. Vangrevelinghe:Novartis: Employment. De Pover:Novartis: Employment. Regnier:Novartis: Employment. Erdmann:Novartis: Employment. Hofmann:Novartis: Employment. Eck:Novartis: Consultancy, Research Funding. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Radimerski:Novartis Pharma AG: Employment. Weinstock:Novartis: Consultancy, Research Funding.

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

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 538-538
Author(s):  
Christopher Peter Mill ◽  
Warren Fiskus ◽  
Dyana T. Saenz ◽  
Bernardo H Lara ◽  
Vrajesh Karkhanis ◽  
...  
Keyword(s):  
Research Funding ◽  
Ex Vivo ◽  
Myeloproliferative Neoplasms ◽  
Mrna Level ◽  
Genetic Alterations ◽  
Cross Resistance ◽  
Oncology Research ◽  
Bet Inhibitors ◽  
Bet Protein ◽  
And Control

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 &gt; 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 &lt; 0.001). Conversely, ectopic overexpression of TCF7L2 or JMJD6 significantly conferred BETi-persister-resistance in HEL and SET2 cells (p &lt; 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 &lt; 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 &lt; 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.

scholarly journals Endothelial Colony-Forming Cells (ECFC) As an Autologous Model for Studying Endothelial Pathophysiology in Sickle Cell Anemia and Myeloproliferative Neoplasms

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 74-74
Author(s):  
Stephanie Ospina-Prieto ◽  
Bruno K L Duarte ◽  
Jessica O F Guanaes ◽  
Fernando F. Costa ◽  
Margareth C Ozelo
Keyword(s):  
Endothelial Cells ◽  
Research Funding ◽  
Progenitor Cell ◽  
Myeloproliferative Neoplasms ◽  
Vascular Diseases ◽  
Cell Marker ◽  
Healthy Individuals ◽  
Network Parameters ◽  
Endothelial Colony Forming Cells

Abstract Background: Endothelial colony-forming cells (ECFC) are an important source of autologous endothelial cells to study its implication in the pathophysiology of diseases with risk of vaso-occlusive events. Currently, our research group began to elucidate the ECFC mechanisms that contribute to the complex clinical vascular manifestations in two diseases, sickle cell anemia (SCA) and myeloproliferative neoplasms (MPN). Aims: In this study, we analyzed functional in vitro assays of endothelial cells. The adhesion to red blood cell (RBC), migration and angiogenesis process of ECFC isolated from patients with SCA and MPN, as well as from healthy individuals (CTR) were evaluated in seeking an expanded understanding of the biology of the endothelial cell and its role in vascular events. Methods: ECFC were obtained through the isolation and culture of human peripheral blood mononuclear cells. ECFC were isolated from 8 patients with SCA under regular transfusion, 6 patients with MPN and 10 CTR. Human umbilical vein endothelial cells (HUVEC) were used as additional controls. Flow cytometry of ECFC indicated that all cells were highly positive for endothelial cell markers CD31, CD144, CD146 and KDR with no indication of CD45 (leukocyte antigens), CD133 (endothelial progenitor cell marker) and CD34 (hematopoietic progenitor cell marker). RBCs from healthy individuals were obtained after centrifugation of whole blood. Cellular adhesion was evaluated after incubation of ECFCs with RBCs in the presence or absence of inflammatory stimulus (TNF-α). Endothelial adhesion molecules were analyzed by flow cytometry (ICAM-1, VCAM-1, E and P-selectin). ECFC migration was assessed using a scratch-wound healing assay and wound regression was analyzed by time-lapse videos. Angiogenesis capacity was evaluated through three-dimensional ECFC cultures in Matrigel. Network parameters (segments, junctions and meshes) were characterized during 24h after seeding. All experiments were performed in triplicate. Results: In total, 48 ECFC colonies were established, 10 from SCA, 25 MPN and 13 CTR. We observed a higher percentage of adhered RBCs to ECFCs isolated from patients with SCA (14.0%) and MPN (23.4%) without TNF-ɑ stimulus, when compared to ECFC from CTR, (8.4%, p< 0.04 and p< 0.001, respectively), and HUVEC (9.4%, p< 0.05 and p< 0.02, respectively). No differences were detected in the expression of adhesion molecules in ECFC. Mean wound regression rates at 14h were 79.9% for SCA, 84.4% for MPN, and 88.8% for CTR. The high variability among colonies in each group could explain why this difference was not statistically significant. Finally, HUVECs had a shorter time for wound closure, with complete wound regression at 10h. The angiogenesis analysis at 15h ECFC from SCA and MPN had, respectively, 20% and 50% less network parameters than CTR. Then beyond 15h post-seeding, the network parameters regressed until reaching a plateau. At 24h the segments began to disappear progressively, leading to a marked reduction in 40h. Unlike ECFC, HUVEC presented a high network formation. Conclusions: Our findings reveal distinct functional properties and behavior between the ECFCs from two diseases with vascular manifestations, SCA and MPN. ECFC do provide access to patient vascular endothelium and enable us to validate the use of these cells as investigative models. In contrast, HUVECs showed a unique behavior, which differed from both diseased and controls ECFCs. This highlights the differences between autologous in vitro and established cell lines as experimental models for vascular diseases. This raises the question of which is the most representative model of human endothelial pathophysiology in vascular diseases. Disclosures Ozelo: BioMarin: Honoraria, Speakers Bureau; Grifols: Honoraria; Novo Nordisk: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Shire: Honoraria, Research Funding, Speakers Bureau; Bioverativ: Honoraria, Research Funding.

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