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THE YING AND YANG OF JAK SIGNALING : LOSS OF USP9X BUFFERS JAK SIGNALING AND ENHANCES SURVIVAL OF CRLF2-JAK-STAT EXPRESSING B CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA.
Author(s): ,
Omer Schwartzman
Affiliations:
The Gene Development and Environment Pediatric Research Institute, Pediatric Hemato-Oncology, Edmond and Lily Safra Children Hospital, Sheba Medical Center,Ramat Gan,Israel;Department of human molecular genetics and biochemistry,Sackler medical school,Tel Aviv University,Tel Aviv,Israel
,
Angela Maria Savino
Affiliations:
The Gene Development and Environment Pediatric Research Institute, Pediatric Hemato-Oncology, Edmond and Lily Safra Children Hospital, Sheba Medical Center,Ramat Gan,Israel;Department of human molecular genetics and biochemistry,Sackler medical school,Tel Aviv University,Tel Aviv,Israel
,
Michael Gombert
Affiliations:
Department of Pediatric Hemato-Oncology and Immunology,Medical Faculty,Heinrich-Heine-University,Dusseldorf,Germany
,
Chiara Palmi
Affiliations:
Centro Ricerca M. Tettamanti, Clinica Pediatrica, Università di Milano Bicocca,Fondazione MBBM/Ospedale San Gerardo,Monza,Italy
,
Gunnar Cario
Affiliations:
Department of Pediatrics,Medical University of Schleswig Holstein,Kiel,Germany
,
Martin Schrappe
Affiliations:
Department of Pediatrics,Medical University of Schleswig Holstein,Kiel,Germany
,
Cornelia Eckert
Affiliations:
Pediatric Oncology/Hematology,Charité - Universitätsmedizin,Berlin,Germany
,
Arend von Stackelberg
Affiliations:
Pediatric Oncology/Hematology,Charité - Universitätsmedizin,Berlin,Germany
,
Jin-Yan Huang
Affiliations:
State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital, Jiao Tong University School of Medicine,Shanghai,China
,
Michal Hameiri-Grossman
Affiliations:
Pediatric Hematology Oncology,Schneider Children's Medical Center of Israel,Petah Tikva,Israel
,
Smadar Avigad
Affiliations:
Pediatric Hematology Oncology,Schneider Children's Medical Center of Israel,Petah Tikva,Israel
,
Geertruy te Kronnie
Affiliations:
Department of Women's and Children's Health,University of Padova,Padova,Italy
,
Ifat Geron
Affiliations:
The Gene Development and Environment Pediatric Research Institute, Pediatric Hemato-Oncology, Edmond and Lily Safra Children Hospital, Sheba Medical Center,Ramat Gan,Israel;Department of human molecular genetics and biochemistry,Sackler medical school,Tel Aviv University,Tel Aviv,Israel
,
Yehudit Birger
Affiliations:
The Gene Development and Environment Pediatric Research Institute, Pediatric Hemato-Oncology, Edmond and Lily Safra Children Hospital, Sheba Medical Center,Ramat Gan,Israel;Department of human molecular genetics and biochemistry,Sackler medical school,Tel Aviv University,Tel Aviv,Israel
,
Avigail Rein
Affiliations:
The Gene Development and Environment Pediatric Research Institute, Pediatric Hemato-Oncology, Edmond and Lily Safra Children Hospital, Sheba Medical Center,Ramat Gan,Israel;Department of human molecular genetics and biochemistry,Sackler medical school,Tel Aviv University,Tel Aviv,Israel
,
Giulia Zarfati
Affiliations:
The Gene Development and Environment Pediatric Research Institute, Pediatric Hemato-Oncology, Edmond and Lily Safra Children Hospital, Sheba Medical Center,Ramat Gan,Israel;Department of human molecular genetics and biochemistry,Sackler medical school,Tel Aviv University,Tel Aviv,Israel
,
Ute Fischer
Affiliations:
Department of Pediatric Hemato-Oncology and Immunology,Medical Faculty,Heinrich-Heine-University,Dusseldorf,Germany
,
Martin Stanulla
Affiliations:
Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germany
,
Andrea Biondi
Affiliations:
Centro Ricerca M. Tettamanti, Clinica Pediatrica, Università di Milano Bicocca,Fondazione MBBM/Ospedale San Gerardo,Monza,Italy
,
Giovanni Cazzaniga
Affiliations:
Centro Ricerca M. Tettamanti, Clinica Pediatrica, Università di Milano Bicocca,Fondazione MBBM/Ospedale San Gerardo,Monza,Italy
,
Bridget Wagner
Affiliations:
Center for the Science of Therapeutics, Proteomics Platform, Medical andPopulation Genetics Program, and ○Howard Hughes Medical Institute, Broad Institute,Cambridge,United States
,
Zhu Chen
Affiliations:
State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital, Jiao Tong University School of Medicine,Shanghai,China
,
Saijuan Chen
Affiliations:
State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital, Jiao Tong University School of Medicine,Shanghai,China
,
Amos Tanay
Affiliations:
Department of Computer Science and Applied Mathematics, and Department ofBiological Regulation,Weizmann Institute of Science,Rehovot,Israel
,
Arndt Borkhardt
Affiliations:
Department of Pediatric Hemato-Oncology and Immunology,Medical Faculty,Heinrich-Heine-University,Dusseldorf,Germany
Shai Izraeli
Affiliations:
The Gene Development and Environment Pediatric Research Institute, Pediatric Hemato-Oncology, Edmond and Lily Safra Children Hospital, Sheba Medical Center,Ramat Gan,Israel;Department of human molecular genetics and biochemistry,Sackler medical school,Tel Aviv University,Tel Aviv,Israel
(Abstract release date: 05/18/17) EHA Library. Schwartzman O. 06/24/17; 181723; S436
Omer Schwartzman
Omer Schwartzman
Contributions
Abstract

Abstract: S436

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 11:30 - 11:45

Location: Room N105

Background

Children with Down syndrome (DS) are prone to development of high risk B cell precursor (BCP) acute lymphoblastic leukemias (DS-ALL) that differ genetically from most sporadic pediatric ALLs. Chromosomal rearrangements causing increased expression of CRLF2, the receptor for thymic stromal lymphopoietin (TSLP), characterize about half of DS-ALLs. 

Aims
Understanding the pathogenesis of relapse of DS-ALL relating to their CRLF2 status.

Methods
Integrative genomic analysis of matched diagnosis remission and relapse DS-ALLs, pharmacological inhibition and genetic CRISPR mediated silencing.

 

Results

Genomic analysis of 25 matched diagnosis remission and relapse DS-ALLs reveled lesions affecting known driver genes in the samples. In 80% of the patients we detected activating mutations in genes whose protein products are involved in signaling, including receptors (CRLF2, IL7R, FLT3), or downstream effector enzymes (JAK1/2, KRAS and NRAS). In contrast to a previous report, we observed that lesions in CRLF2 are early events during DS-ALL evolution, as is evident by its high allelic frequency, and are maintained at relapse. The genetic make-up differed significantly between these two major subtypes of DS-ALLs. CRLF2neg DSALLs were characterized by enhanced RAS signaling coupled by mutations in chromatin remodeling genes, in particular CREBBP. In contrast CRLF2pos DS-ALLs were characterized by high dynamics of proliferative signaling. At diagnosis CRLF2 rearrangements were almost always combined with secondary activating signaling events in JAK-STAT pathway suggesting that signaling is driving the development of these leukemias. However JAK2 mutations were often lost at relapse and replaced by clones with mutated RAS. Thus the presence of JAK2 activating mutations at the time of diagnosis are associated with sensitivity to upfront chemotherapy. Surprisingly we discovered loss-of-function mutations or deletions of USP9X, a deubiquitinase previously described as an oncogene that positively regulates JAK2 signaling, in 25% of CRLF2pos ALLs, in both our study and published data,.  We therefore tested the counterintuitive hypothesis that loss of a positive regulator of JAK2 enhances the fitness of JAK-STAT driven JAK2 mutated leukemic cells. Both pharmacological inhibition and genetic CRISPR mediated silencing of USP9X reduced STAT5 phosphorylation and enhanced the survival of CRLF2-JAK2R683Gs transduced ALL cells.  To test directly the effect of JAK inhibition, we treated CRLF2/JAK2R683G transduced cells with increasing doses of ruxolitinib, a JAK inhibitor currently in clinical trials for CRLF2-JAK-STAT ALLs. Strikingly while high doses (>2 μM) were cytotoxic, low doses (0.25 μM) enhanced the survival of CRLF2-JAK2R683Gs expressing ALL cells. 

Conclusion

These observations suggest that genetic or pharmacological restraining of JAK-STAT signaling may be beneficial to leukemic B cell precursors by enhancing the fitness of JAK-STAT “driven” ALL. This and the reduction of JAK-mutated clones at relapse suggest that the therapeutic effect of JAK2 specific inhibitors may be limited. Rather, combined signaling inhibitors or direct targeting of the TSLP receptor may be a useful therapeutic strategy for DS-ALL.

Session topic: 1. Acute lymphoblastic leukemia - Biology

Keyword(s): Signaling, Down Syndrome, Acute lymphoblastic leukemia

Abstract: S436

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 11:30 - 11:45

Location: Room N105

Background

Children with Down syndrome (DS) are prone to development of high risk B cell precursor (BCP) acute lymphoblastic leukemias (DS-ALL) that differ genetically from most sporadic pediatric ALLs. Chromosomal rearrangements causing increased expression of CRLF2, the receptor for thymic stromal lymphopoietin (TSLP), characterize about half of DS-ALLs. 

Aims
Understanding the pathogenesis of relapse of DS-ALL relating to their CRLF2 status.

Methods
Integrative genomic analysis of matched diagnosis remission and relapse DS-ALLs, pharmacological inhibition and genetic CRISPR mediated silencing.

 

Results

Genomic analysis of 25 matched diagnosis remission and relapse DS-ALLs reveled lesions affecting known driver genes in the samples. In 80% of the patients we detected activating mutations in genes whose protein products are involved in signaling, including receptors (CRLF2, IL7R, FLT3), or downstream effector enzymes (JAK1/2, KRAS and NRAS). In contrast to a previous report, we observed that lesions in CRLF2 are early events during DS-ALL evolution, as is evident by its high allelic frequency, and are maintained at relapse. The genetic make-up differed significantly between these two major subtypes of DS-ALLs. CRLF2neg DSALLs were characterized by enhanced RAS signaling coupled by mutations in chromatin remodeling genes, in particular CREBBP. In contrast CRLF2pos DS-ALLs were characterized by high dynamics of proliferative signaling. At diagnosis CRLF2 rearrangements were almost always combined with secondary activating signaling events in JAK-STAT pathway suggesting that signaling is driving the development of these leukemias. However JAK2 mutations were often lost at relapse and replaced by clones with mutated RAS. Thus the presence of JAK2 activating mutations at the time of diagnosis are associated with sensitivity to upfront chemotherapy. Surprisingly we discovered loss-of-function mutations or deletions of USP9X, a deubiquitinase previously described as an oncogene that positively regulates JAK2 signaling, in 25% of CRLF2pos ALLs, in both our study and published data,.  We therefore tested the counterintuitive hypothesis that loss of a positive regulator of JAK2 enhances the fitness of JAK-STAT driven JAK2 mutated leukemic cells. Both pharmacological inhibition and genetic CRISPR mediated silencing of USP9X reduced STAT5 phosphorylation and enhanced the survival of CRLF2-JAK2R683Gs transduced ALL cells.  To test directly the effect of JAK inhibition, we treated CRLF2/JAK2R683G transduced cells with increasing doses of ruxolitinib, a JAK inhibitor currently in clinical trials for CRLF2-JAK-STAT ALLs. Strikingly while high doses (>2 μM) were cytotoxic, low doses (0.25 μM) enhanced the survival of CRLF2-JAK2R683Gs expressing ALL cells. 

Conclusion

These observations suggest that genetic or pharmacological restraining of JAK-STAT signaling may be beneficial to leukemic B cell precursors by enhancing the fitness of JAK-STAT “driven” ALL. This and the reduction of JAK-mutated clones at relapse suggest that the therapeutic effect of JAK2 specific inhibitors may be limited. Rather, combined signaling inhibitors or direct targeting of the TSLP receptor may be a useful therapeutic strategy for DS-ALL.

Session topic: 1. Acute lymphoblastic leukemia - Biology

Keyword(s): Signaling, Down Syndrome, Acute lymphoblastic leukemia

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