Genetic Resistance to JAK2 Enzymatic Inhibitors Is Overcome by HSP90 Inhibition

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.

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Genetic resistance to JAK2 enzymatic inhibitors is overcome by HSP90 inhibition

Journal of Experimental Medicine ◽

10.1084/jem.20111694 ◽

2012 ◽

Vol 209 (2) ◽

pp. 259-273 ◽

Cited By ~ 113

Author(s):

Oliver Weigert ◽

Andrew A. Lane ◽

Liat Bird ◽

Nadja Kopp ◽

Bjoern Chapuy ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Lymphoblastic Leukemia ◽

Genetic Resistance ◽

Heat Shock Protein 90 ◽

Jak2 V617f ◽

Kinase Domain ◽

Hsp90 Inhibitor ◽

Janus Kinase ◽

Cytokine Receptor ◽

Hsp90 Inhibition

Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of myeloproliferative neoplasms (MPNs), B cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit cytokine receptor–like factor 2 (CRLF2), and other tumors with constitutive JAK2 signaling. In this study, we identify G935R, Y931C, and E864K mutations within the JAK2 kinase domain that confer resistance across a panel of JAK inhibitors, whether present in cis with JAK2 V617F (observed in MPNs) or JAK2 R683G (observed in B-ALL). G935R, Y931C, and E864K do not reduce the sensitivity of JAK2-dependent cells to inhibitors of heat shock protein 90 (HSP90), which promote the degradation of both wild-type and mutant JAK2. HSP90 inhibitors were 100–1,000-fold more potent against CRLF2-rearranged B-ALL cells, which correlated with JAK2 degradation and more extensive blockade of JAK2/STAT5, MAP kinase, and AKT signaling. In addition, the HSP90 inhibitor AUY922 prolonged survival of mice xenografted with primary human CRLF2-rearranged B-ALL further than an enzymatic JAK2 inhibitor. Thus, HSP90 is a promising therapeutic target in JAK2-driven cancers, including those with genetic resistance to JAK enzymatic inhibitors.

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Ultrasensitive Duplex Sequencing of Pretreatment ABL1 Kinase Domain Mutations in Patients with Newly Diagnosed Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2018-99-115369 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1548-1548 ◽

Cited By ~ 2

Author(s):

Nicholas J Short ◽

Hagop M. Kantarjian ◽

Koji Sasaki ◽

Farhad Ravandi ◽

Jorge E. Cortes ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Kinase Domain ◽

Newly Diagnosed ◽

Resistance Mutations ◽

Genetics Research ◽

Duplex Sequencing ◽

Philadelphia Chromosome Positive

Abstract Background: Kinase domain (KD) mutations in ABL1 are the dominant mechanism of relapse in patients (pts) with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). Previous studies in Ph+ ALL have suggested that some pts harbor subclonal T315I mutations prior to tyrosine kinase inhibitor (TKI) treatment. However, nearly all reports have relied upon inherently error prone RT-PCR to generate template cDNA prior to mutation analysis. We hypothesized that conventional assays might over-estimate the incidence of pre-existing resistance mutations, and that improved sequencing accuracy might yield important information for risk stratification and TKI selection. Methods: Duplex sequencing (DS) is a molecular tagging method that improves the accuracy of conventional next-generation sequencing by more than 10,000-fold, by comparing the nucleotide sequences of each strand of double-stranded molecules. ABL1 DS was performed on genomic DNA from 64 pts with newly diagnosed Ph+ ALL treated with hyper-CVAD plus a TKI. DS of exons 4-10 of ABL1 was performed to an average molecular depth of >10,000x. Among pts who relapsed, using RNA extracted from relapse samples, the KD (codons 221 through 500) of the BCR-ABL1 fusion transcript was sequenced by the Sanger method using a nested PCR approach, with a detection limit of 10-20%. Results: The median age of the cohort was 54 years (range, 20-80 years). The TKI used was imatinib in 5 pts, dasatinib in 38 pts, and ponatinib in 21 pts. All pts achieved complete remission, and 12 pts (19%) underwent allogeneic stem cell transplantation in first remission. A total of 115 pretreatment ABL1 KD mutations were detected among 47 pts (73%). The median number of pretreatment ABL1 KD mutations was 2 (range, 0-6 mutations). The median variant allelic frequency (VAF) of the detected somatic mutations was 0.008% (range, 0.004%-0.649%). Five mutations (4%) and 40 mutations (35%) were present at a VAF ≥0.1% and ≥0.01%, respectively. Eleven mutations known to confer resistance to at least one TKI were detected in 7 pts (11%), and included: F317L in 4 pts, E225K in 2 pts, and E225V, L384M, M244V, Q252H and T315I in 1 pt each. Five mutations were detected in ≥1 pretreatment sample (F317L in 4 pts, M244V in 3 pts, and E255K, E459K and V355V in 2 pts each). Of these 7 pts with pretreatment resistance mutations, 5 pts received a TKI known not to be sensitive to the mutation(s); 2 pts who received ponatinib had mutations at least intermediately sensitive to ponatinib (1 pt with F317L and 1 pt with both E255K and M244V mutations). With a median duration of follow-up of 54 months (range, 1-124 months), 18 pts have relapsed. None of the 7 pts with known pretreatment resistance mutations relapsed. There was no difference in the number of pretreatment mutations between pts who relapsed and those who did not (median mutations: 1 [range, 0-4] and 2 [range, 0-6], respectively; P=0.26). Of the 18 pts who relapsed, 14 underwent Sanger sequencing for ABL1 KD mutations at the time of relapse. Clonal resistance mutations were detected at relapse in 9 pts (64% of sequenced samples): T315I in 6 pts, and F317I, V229L and V338G in 1 pt each (all in pts treated with dasatinib, except V338G in pt on imatinib). Relapse mutations were not observed in pretreatment samples in any of the pts. Conclusions: DS identified very low level pretreatment ABL1 KD mutations in a majority of pts with newly diagnosed Ph+ ALL but these appear inconsequential. Ninety percent of mutations identified have not been described as resistance mutations, suggesting that they may be either synonymous or functionally neutral amino acid changes resulting from normal aging. There was no apparent association of these mutations and risk of relapse, even in the minority of cases in which known resistance mutations were detected at baseline. These data suggest that pretreatment testing for ABL1 KD mutations in Ph+ ALL is unlikely to affect treatment decisions. Disclosures Short: Takeda Oncology: Consultancy. Sasaki:Otsuka Pharmaceutical: Honoraria. Ravandi:Jazz: Honoraria; Orsenix: Honoraria; Orsenix: Honoraria; Seattle Genetics: Research Funding; Sunesis: Honoraria; Xencor: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Abbvie: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Macrogenix: Honoraria, Research Funding; Jazz: Honoraria; Abbvie: Research Funding; Xencor: Research Funding; Macrogenix: Honoraria, Research Funding; Sunesis: Honoraria. Cortes:Pfizer: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Arog: Research Funding. Konopleva:Stemline Therapeutics: Research Funding. Radich:TwinStrand Biosciences: Research Funding. Jabbour:Takeda: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis: Research Funding; Pfizer: Consultancy, Research Funding; Abbvie: Research Funding.

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Mutation in Abl kinase with altered drug-binding kinetics indicates a novel mechanism of imatinib resistance

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2111451118 ◽

2021 ◽

Vol 118 (46) ◽

pp. e2111451118

Author(s):

Agatha Lyczek ◽

Benedict-Tilman Berger ◽

Aziz M. Rangwala ◽

YiTing Paung ◽

Jessica Tom ◽

...

Keyword(s):

Binding Site ◽

Kinase Domain ◽

Protein Kinase Inhibitors ◽

Drug Binding ◽

The Body ◽

Binding Kinetics ◽

Wild Type ◽

Resistance Mutations ◽

Imatinib Resistance ◽

Abl Kinase

Protein kinase inhibitors are potent anticancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for chronic myeloid leukemia by 80%, but 22 to 41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, where more than a hundred different mutations have been identified. Some mutations are located near the imatinib-binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug-binding site, and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies. Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease relevant or resistance causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than twofold compared to Abl wild type. Surprisingly, one-third of mutations in the Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified three clinical Abl mutations that bind imatinib with wild type–like affinity but dissociate from imatinib considerably faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the nonequilibrium environment of the body where drug export and clearance play critical roles.

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Patterns of Care and Outcomes Among Older Patients with Myelofibrosis: A Population-Based Study

Blood ◽

10.1182/blood-2020-137543 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 21-22

Author(s):

Shelby Meckstroth ◽

Rong Wang ◽

Xiaomei Ma ◽

Nikolai A. Podoltsev

Keyword(s):

Median Survival ◽

Research Funding ◽

Multivariable Analysis ◽

Population Based ◽

Janus Kinase ◽

Patterns Of Care ◽

Age At Diagnosis ◽

Jak Inhibitor ◽

Risk Of Death

Background: Myelofibrosis (MF) is a Philadelphia chromosome negative myeloproliferative neoplasm associated with systemic and splenomegaly-related symptoms, cytopenias and decreased survival. Approval of ruxolitinib, an oral janus kinase (JAK)-inhibitor, for higher-risk MF patients (pts) by the Food and Drug Administration in 11/ 2011 opened a new era of targeted treatment for this disease. There are limited data on the "real-world" clinical experiences and outcomes of pts with MF treated in the JAK inhibitor era. MF became reportable to population-based cancer registries including the Surveillance, Epidemiology and End Results (SEER) program in 2001, making its investigation possible at the population level. The objective of this study was to assess the patterns of care and outcomes of older MF pts in the ruxolitinib era. Methods: Using the linked SEER-Medicare database, we identified a cohort of older pts diagnosed with MF from 2007 through 2015 who fulfilled the following eligibility criteria: 1) aged 66-99 years at diagnosis; 2) had known month of diagnosis; 3) were not identified from death certificate or autopsy only; 4) had continuous enrollment in Medicare Parts A, B and no enrollment in health maintenance organizations from 1 year before diagnosis until the end of follow-up (death or 12/31/2016, whichever came first); 5) had continuous enrollment in Medicare Part D from diagnosis until the end of follow-up; and 6) bone marrow biopsy claim from 1 year before diagnosis to end of follow up. Treatments were assessed via Medicare parts B&D claims. Kaplan-Meier curves and log-rank tests were used to compare survival between patient groups. Multivariable cox proportional hazards regression models were used to assess the effect of ruxolitinib use on survival in MF pts. Aside from treatment, we considered the influence of several characteristics on survival, including age at diagnosis, sex, race/ethnicity, marital status, comorbidities, SEER region and percentage living in poverty at the census tract level. Results: Among 528 MF pts, median age at diagnosis was 76 (interquartile range [IQR], 71- 80) years with 88.8% white and 56.1% male. 230 pts were diagnosed in the early era (2007-2011), and 298 in the late era (2012-2015), of which 113 (37.9%) were ruxolitinib users. There was no difference among any evaluated characteristics between two eras and by ruxolitinib status in the late era. The median duration of ruxolitinib use was 11.9 months. Similar number of pts started at 5, 10, 15 and 20 mg twice a day (BID) (Figure 1). Among 31 pts who started at ≤5 mg BID, 15 (48.4%) never had their dose of ruxolitinib escalated. While on ruxolitinib treatment, nearly half of the pts received additional medications for symptom management including hydroxyurea (22.6%), prednisone (17.9%) or both (10.4%). < 11 users were able to go up to the highest dose of 25 mg BID. Ruxolitinib was interrupted > 30 days for 31 times by 20 of 113 (17.7%) pts with median interruption duration of 43 (IQR 34-71) days. The median survival was 2.70 (95% confidence interval [CI]: 1.87-3.41) years and 2.62 (95% CI: 2.15-3.07) for the early and late era pts, respectively (p for log-rank 0.91). The multivariable analysis showed no impact of diagnosis era on survival (late vs early era hazard ratio (HR) of 1.08, 95% CI 0.83-1.40; p= 0.57). There was no difference in survival by ruxolitinib status (log-rank test, p=0.31), with a median survival of 2.76 (95% CI: 2.01-4.15) years and 2.53 (95% CI: 1.92-3.07) years among users and non-users, respectively (Figure 3). In the multivariable analysis, the risk of death among ruxolitinib users compared to non-users was not statistically significant with HR of 0.82 (95% CI 0. 59-1.16; p= 0.26). Conclusions: Older MF pts treated with ruxolitinib had similar survival when compared to pts who did not receive this medication, but the choice of ruxolitinib might have been influenced by disease risk which we were unable to assess. For many ruxolitinib users, the drug was interrupted, the dose was not escalated, additional medications were used concurrently (possibly to help control disease manifestation), and treatment was discontinued quickly after initiation. Optimization of ruxolitinib use may be necessary to accomplish better outcomes. Furthermore, development of new drugs which may be used together with ruxolitinib or after its discontinuation is needed. The work was supported by The Frederick A. Deluca Foundation. Disclosures Wang: Celgene/BMS: Research Funding. Ma:Celgene/BMS: Research Funding; BMS: Consultancy. Podoltsev:Jazz Pharmaceuticals: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Agios Pharmaceuticals: Consultancy, Honoraria; Sunesis Pharmaceuticals: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Blueprint Medicines: Consultancy, Honoraria; Bristol-Myers Squib: Consultancy, Honoraria; Genentech: Research Funding; AI Therapeutics: Research Funding; Samus Therapeutics: Research Funding; Astellas Pharma: Research Funding; Kartos Therapeutics: Research Funding; CTI biopharma: Consultancy, Honoraria, Research Funding; Boehringer Ingelheim: Research Funding; Novartis: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Astex Pharmaceuticals: Research Funding; Daiichi Sankyo: Research Funding; Arog Pharmaceuticals: Research Funding.

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STAT5 is Expressed in CD34+/CD38− Stem Cells and Serves as a Potential Molecular Target in Ph-Negative Myeloproliferative Neoplasms

Cancers ◽

10.3390/cancers12041021 ◽

2020 ◽

Vol 12 (4) ◽

pp. 1021 ◽

Cited By ~ 3

Author(s):

Emir Hadzijusufovic ◽

Alexandra Keller ◽

Daniela Berger ◽

Georg Greiner ◽

Bettina Wingelhofer ◽

...

Keyword(s):

Stem Cells ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Janus Kinase ◽

Signaling Cascades ◽

Nuclear Compartment ◽

Downstream Signaling ◽

Direct Targeting ◽

Calr Mutations

Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34+/CD38− MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34+/CD38− MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.

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To Study Resistant Mechanisms and Reversal In An Imatinib Resistant Ph+ Positive Acute Lymphoblastic Leukemia Cell Line

Blood ◽

10.1182/blood.v116.21.2135.2135 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2135-2135

Author(s):

Hongyun Xing ◽

Yuping Gong ◽

Ting Liu

Keyword(s):

Cell Signaling ◽

Cell Line ◽

Lymphoblastic Leukemia ◽

Kinase Domain ◽

Rt Pcr ◽

Imatinib Resistance ◽

Abl Kinase ◽

Imatinib Resistant ◽

Cell Signaling Pathways

Abstract Abstract 2135 Objective To establish an imatinib resistant Bcr-Abl positive acute lymphoblastic leukemia (ALL) cell line in vitro and to study imatibin resistance in Ph+ ALL. The reversal of the imatinib resistance by rapamycin, the second generation tyrosine kinase inhibitor and proteasome inhibitor was studied. Methods Ph(+) ALL SUP-B15 cell line was cultured in gradually increasing concentrations of imatinib to generate the imatinib resistant cell line at 6 μM imatinib. The cytotoxic effect of imatinib and other drugs was analyzed by MTT assay. RT-PCR, flow cytometry, Western blot analyses of proteins, DNA sequence analysis of ABL kinase domain were used to clarify the possible mechanisms of the imatinib resistance in the SUP-B15/RI cell line. Results We established the imatinib resistant Ph+ ALL cell line. The fusion bcr-abl gene was 6.1 times as high as that of the parental sensitive cell, and the mdr1 gene also increased 1.7 times in SUP-B15/RI cell line by the RT-PCR detection. However, the expression of hoct1 Abcl–2 and topoIIα gene were no difference between two cell lines by the RT-PCR detection. A K362S point mutation in the Abl kinase domain of SUP-B15/RI was found. The detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κBA JNK and STAT showed the expression of PTEN and 4EBP-1 was down-regulated, AKT, mTOR and P70S6K was up-regulated and the expression of other cell signaling pathways in SUP-B15/RI was similar to its parental sensitive cell line. Dasatinib, nilotinib, and bortezomib could inhibit proliferation of SUP-B15/RI cells at nM concentration. SUP-B15/RI cell line also showed partial resistance to dasatinib and nilotinib, but not bortezomib. The combination of imatinib with rapamycin had synergistic effect to the resistance cell line. Conclusion In vitro, we establish imatinib resistant Ph + ALL cell line. Overexpression of bcr-abl and mdr1 gene, K362S point mutation in ABL kinase domain and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line were involved in the resistance mechanisms. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinaeses dasatinib and nilotinib,not bortezomib in vitro. However, the combination of imatinib with rapamycin can partially overcome the resistance. Blockade of the ubiquitin-proteasome could be a promising pathway to overcome resistance to imatinib. Disclosures: No relevant conflicts of interest to declare.

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Direct Activation of STAT5 by TEL-Lyn Fusion Protein Promotes Induction of Myeloproliferative Neoplasms with Myelofibrosis

Blood ◽

10.1182/blood.v116.21.4114.4114 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4114-4114

Author(s):

Yusuke Takeda ◽

Chiaki Nakaseko ◽

Hiroaki Tanaka ◽

Masahiro Takeuchi ◽

Makiko Yui ◽

...

Keyword(s):

Bone Marrow ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Signaling Molecules ◽

Janus Kinase ◽

Idiopathic Myelofibrosis ◽

Lyn Kinase

Abstract Abstract 4114 Background Myeloproliferative neoplasms (MPN), a group of hematopoietic stem cell (HSC) disorders, are often accompanied by myelofibrosis. The V617F somatic mutation in the Janus kinase 2 (JAK2) gene has recently been found in the majority of patients with polycythemia vera (PV) and more than half of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). The expression of JAK2 V617F causes a PV-like disease with myelofibrosis in a murine bone marrow (BM) transplant model. In addition, a gain-of-function c-MPL W515 mutation was described in nearly 10% of patients with JAK2 V617F-negative IMF. However, the mechanism responsible for MPD and the formation of myelofibrosis in patients without JAK2 or c-MPL mutations is still unclear. We previously identified the fusion of the TEL gene to the Lyn gene (TEL-Lyn) in idiopathic myelofibrosis with ins(12;8)(p13;q11q21). The introduction of TEL-Lyn into HSCs resulted in fatal MPN with massive myelofibrosis in mice, implicating the rearranged Lyn kinase in the pathogenesis of MPN with myelofibrosis. However, the signaling molecules directly downstream from and activated by TEL-Lyn remain unknown. Design and Methods We examined the signaling pathways activated by TEL-Lyn by Western blotting, immunoprecipitation, and in vitro kinase assay using a TEL-Lyn kinase-dead mutant as a control. We further characterized the functional properties of Stat5-deficient HSCs transduced with TEL-Lyn by colony-forming assay and bone marrow transplantation to evaluate the role of STAT5 in TEL-Lyn-induced MPN. Results TEL-Lyn was demonstrated to be constitutively active as a kinase through autophosphorylation. In TEL-Lyn-expressing cells, STAT5, STAT3, and Akt were constitutively activated. Among these signaling molecules, STAT5 was activated most prominently and this occurred without the activation of Jak2, the major kinase for STAT5. TEL-Lyn was co-immunoprecipitated with STAT5, and STAT5 was phosphorylated when incubated with TEL-Lyn, but not with TEL-Lyn kinase-dead mutant. These results indicate that TEL-Lyn interacts with STAT5 and directly activates STAT5 both in vitro and in vivo. Of note, the capacity of TEL-Lyn to support the formation of hematopoietic colonies under cytokine-free conditions in vitro and to induce MPN with myelofibrosis in vivo was profoundly attenuated in a Stat5-null background. Conclusions In this study, we clearly showed that TEL-Lyn directly activates STAT5 and the capacity of TEL-Lyn to induce MPN with myelofibrosis was profoundly attenuated in the absence of STAT5. Our findings of TEL-Lyn in this study support the role of the Src family kinases in the regulation of STAT pathways and implicate active Lyn in the alternative pathway for STAT activation in pathological cytokine signaling. Our mouse model of MPD with myelofibrosis would be beneficial for the analysis of therapeutic approaches for myelofibrosis. Disclosures: No relevant conflicts of interest to declare.

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Treating Ph+ Acute Lymphoblastic Leukemia (ALL) in the Elderly: The Sequence of Two Tyrosine Kinase Inhibitors (TKI) (Nilotinib and Imatinib) Does Not Prevent Mutations and Relapse.

Blood ◽

10.1182/blood.v120.21.2601.2601 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2601-2601 ◽

Cited By ~ 4

Author(s):

Cristina Papayannidis ◽

Paola Fazi ◽

Alfonso Piciocchi ◽

Francesco Di Raimondo ◽

Giovanni Pizzolo ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Tyrosine Kinase ◽

Research Funding ◽

Kinase Inhibitors ◽

Lymphoblastic Leukemia ◽

The Elderly ◽

Kinase Domain ◽

Intensive Chemotherapy ◽

Molecular Responses ◽

Abl Kinase

Abstract Abstract 2601 Background: Tyrosine Kinase Inhibitors (TKI) have been shown to be very effective for the treatment of Acute Lymphoblastic Leukemia (ALL), with a Complete Hematologic Remission (CHR) rate close to 100%, and a high rate of Complete Cytogenetic and Molecular responses (CCgR and CMR). However, when they are used alone, as single agents, most patients relapse, so that they are currently used in combination with chemotherapy and as a preparation to allogeneic stem cell transplantation (SCT). Since Ph+ ALL is more frequent in the elderly, many patients cannot tolerate intensive chemotherapy and are not eligible for SCT. We have explored if the administration of two TKIs, Nilotinib (NIL) and Imatinib (IM) can improve the results without increasing the toxicity. Aims: To evaluate the response and the outcome of Ph+ ALL patients treated with the sequential administration of NIL and IM, to investigate the type and number of BCR-ABL kinase domain mutations developing during and after the study. Methods: We have designed a study (ClinicalTrials.gov. NCT01025505) in which patients more than 60 years old or unfit for intensive chemotherapy and SCT where treated with two TKIs, NIL 400 mg twice daily, and IM 300 mg twice daily, alternating for 6 weeks for a minimum of 24 weeks (study core) and indefinitely in case of response. The 6-weeks rotation schedule was respected, irrespectively of temporary discontinuations. The primary end-point was the rate of Disease Free Survival (DFS) at 24 weeks (4 courses of treatment); the secondary end points included the evaluation of CHR, CCgR and CMR rates. Mutation analysis was performed by nested RT-PCR amplification of the ABL kinase domain of the BCR-ABL transcript (codons 206 through 421). Amplified products were screened by denaturing-high performance liquid chromatography (D-HPLC). Samples scored positive for the presence of sequence variations were then subjected to direct automatic sequencing to characterize the mutation. Results: 39 patients have been enrolled in 15 Italian hematologic Centers (median age 66 years, range 28–84). Among these, 8 patients were unfit for standard chemotherapy or SCT (median age 50 years, range 28–59). 27 patients were p190, 5 were p210 and 7 were p190/p210. After 6 weeks of treatment, 36 patients were evaluable for response: 34 were in CHR (94%) and 2 in PHR (6%). 23 patients have already completed the study core (24 weeks), 87% were in CHR and 17 are currently continuing therapy in the protocol extension phase. Thus, the OS at 1 year is 79%, and 64% at 2 years. Overall, 1 patient was primarily resistant and 13 patients have relapsed, with a median time to relapse of 7.6 months (range 0.8–16.1 months), for a DFS of 51.3% at 12 months (Figure 1). Mutations detected were T315I in 2 cases, Y253H in 3 cases, T315I and Y253H in 1 case, E255K in 1 case, T315I and E255K in 1 case, E255V and Y253H in 1 case. Two patients were WT. A detailed kinetics of Molecular responses is shown in Table 1. Data on mutational analysis are reported in Table 2. Further details about Cytogenetic and Molecular responses, and about Adverse Events will be provided on site. Conclusions: In this small cohort of Ph+ ALL elderly/unfit patients, the rates of relapse and progression were not likely to be different from the rates observed with Imatinib alone (Vignetti et al, Blood 2007, May 1;109(9):3676-8) and Dasatinib alone (Foà, Blood 2011, Dec 15;118(25):6521-8). It's important to notice that the mutations that occurred at the time of relapse were sensitive to other TKIs (Dasatinib and Ponatinib). Acknowledgments: COFIN, Bologna University, BolognAIL, PRIN, Fondazione del Monte di Bologna e Ravenna, INPDAP. Disclosures: Pizzolo: Hoffmann-La Roche: Consultancy, Honoraria. Luppi:CELGENE CORPORATION: Research Funding. Vallisa:CELGENE CORPORATION: Research Funding. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau.

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Dissecting the Complexity of Philadelphia-Positive Mutated Populations by Ultra-Deep Sequencing of the Bcr-Abl Kinase Domain: Biological and Clinical Implications

Blood ◽

10.1182/blood.v120.21.692.692 ◽

2012 ◽

Vol 120 (21) ◽

pp. 692-692 ◽

Cited By ~ 1

Author(s):

Simona Soverini ◽

Caterina De Benedittis ◽

Katerina Machova Polakova ◽

Adela Brouckova ◽

Fausto Castagnetti ◽

...

Keyword(s):

Deep Sequencing ◽

Research Funding ◽

Kinase Inhibitor ◽

Lymphoblastic Leukemia ◽

Kinase Domain ◽

Mutation Status ◽

Abl Kinase ◽

Selection Of ◽

Higher Sensitivity

Abstract Abstract 692 Background and Aims: In chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (ALL), tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant Bcr-Abl mutants. Mutation status of resistant patients is usually investigated by Sanger sequencing (SS) of the Bcr-Abl kinase domain (KD). Novel ultra-deep sequencing (UDS) technologies allow to conjugate higher sensitivity with the unprecedented possibility to perform instant cloning of thousands of DNA molecules. We thus decided to take advantage of an UDS-based approach in order to: Methods: We retrospectively performed a longitudinal analysis of a total of 111 samples from 35 CML or Ph+ ALL patients who had received sequential treatment with multiple TKIs (two to four TKIs among imatinib, dasatinib, nilotinib, ponatinib) and had experienced sequential relapses accompanied by selection of TKI-resistant mutations. All samples had already been scored by SS; 74/111 (67%) were positive for one (n=33) or multiple (n=41) mutations. UDS of the Bcr-Abl KD was done using Roche 454 technology. UDS allowed to achieve a lower detection limit of at least 0.1% – as compared to 20% of SS. Results: Bcr-Abl KD mutation status was found to be more complex than SS had previously shown in 85/111 (77%) samples (representative examples are detailed in Table 1). In 33/74 (44%) samples known to harbour one or more mutations by SS, UDS revealed that up to four ‘minor’ mutations with 1–20% abundance were present in addition to the ‘dominant’ one(s). The type of mutations could easily be accounted for by TKI exposure history, since the majority were known to be poorly sensitive either to the current or to the previous TKI received. The higher degree of complexity was evident also when the clonal relationships of multiple mutations were reconstructed (Table 1). This revealed that identical mutations may be acquired in parallel by independent populations (e.g., one wild-type and one already harboring a mutation), via the same or different nucleotide changes leading to the same amino acid substitution (convergent evolution). In addition, longitudinal quantitative follow-up of mutated populations revealed that: Conclusions: Disclosures: Soverini: ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Castagnetti:Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Luppi:CELGENE CORPORATION: Research Funding. Rosti:Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

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Germline JAK2 Mutations In The Kinase Domain Are Responsible For Hereditary Thrombocytosis and Are Resistant To JAK2 and HSP90 Inhibitors

Blood ◽

10.1182/blood.v122.21.1603.1603 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1603-1603

Author(s):

Caroline Marty ◽

Cecile Saint-martin ◽

Christian Pecquet ◽

Sarah Grosjean ◽

Joseph Saliba ◽

...

Keyword(s):

Conflicts Of Interest ◽

Jak2 V617f ◽

Surface Expression ◽

Germline Mutations ◽

Kinase Domain ◽

Cell Surface Expression ◽

Hsp90 Inhibitors ◽

Activating Mutations ◽

Signaling Axis ◽

In Cis

Abstract The main molecular basis of essential thrombocytemia and hereditary thrombocytosis are acquired and germline activating mutations affecting the thrombopoietin signaling axis, respectively. We have identified two families with hereditary thrombocytosis presenting novel heterozygous germline mutations of JAK2. One family carries the JAK2 R867Q mutation located in the kinase domain, while the other presents two JAK2 mutations, S755R/R938Q, located in cis in both the pseudokinase and kinase domains. Expression of JAK2 R867Q and S755R/R938Q induced spontaneous growth of Ba/F3-MPL but not of Ba/F3-EPOR cells. Interestingly, both Ba/F3-MPL cells expressing the mutants and platelets from patients displayed a TPO-independent phosphorylation of STAT1. JAK2 R867Q and S755R/R938Q, compared to JAK2 V617F, had significantly longer half-lives correlating with an increased MPL cell surface expression. Moreover, these mutations conferred a resistance to JAK2 and HSP90 inhibitors compared to JAK2 V617F. Our results suggest that the mutations in the kinase domain of JAK2 may confer a weak activation of signaling specifically dependent on MPL, while inducing resistance to clinically available JAK2 inhibitors. Disclosures: No relevant conflicts of interest to declare.

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