SRSF2-P95Hdelays Myelofibrosis Development through Altered JAK/STAT Signaling in JAK2-V617F Megakaryocytes

Abstract Background: The gain-of-function JAK2 V617F mutant is the most common driver mutation identified in myeloproliferative neoplasms (MPNs). Additional somatic variants, also found in other malignant hemopathies, are detected in primary myelofibrosis (MF) and supposed to contribute to fibrosis or leukemia development. One of these mutations affects SRSF2, a gene encoding a component of the splicing machinery. SRSF2 heterozygous mutation mainly affects the proline 95 residue of the protein. Its association with JAK2 V617F correlates with a reduced leukemia free survival. Whether and how SRSF2 P95 variants could favor fibrosis development in JAK2 V617F cells remained unknown. Methods & Results: To investigate how homozygous Jak2 V617F and heterozygous Srsf2 P95H could interact in the hematopoietic tissue, we generated conditional knock-in mice in which the CreERt recombinase expression was driven by the HSC-Scl promoter leading to Jak2 V617F and/or Srsf2 P95H hematopoietic-specific expression upon tamoxifen induction. Srsf2 P95H mutation initially exhibited limited effect on Jak2 V617F-induced polycythemia vera (PV) only slightly reducing erythrocytosis and leukocytosis (through a previously described decrease in B220 + B cell number). The expansion of hematopoietic stem cells (SLAM), multipotent progenitors (MPP) and megakaryocyte progenitors (MKP) observed in Jak2 V617F mice was not affected by Srsf2 status. However, while platelet count was decreasing in Jak2 V617F alone mice at later time point due to fibrosis development, Srsf2 P95H/Jak2 V617F combination further increased platelet counts correlating with a significant delay in the development of myelofibrosis. Bone marrow cells (BM) were transplanted into lethally irradiated recipient mice together with GFP-positive wild-type competitor cells and tamoxifen was administrated after transplantation. Double mutant cells initially demonstrated a limited competitive advantage over wild-type cells as compared to Jak2 V617F-only cells. However, serial transplantation revealed a rapid exhaustion of Jak2 V617F single mutant cells leading to lethal pancytopenia, which was not observed in animals transplanted with Jak2 V617F/Srsf2 P95H double mutant cells. As both monocytes and megakaryocytes (MK) were involved in fibrosis development, we further explored the role of these two cell populations. Spectral flow analysis of monocyte subsets in peripheral blood and BM failed to detect any significant change in double compared to single mutant animals. In contrast, double-mutant mice presented a significant delay in MK maturation with normalized expression of c-Mpl and ploidy. Using mass cytometry, we found ex vivo a higher proportion of MKP and MK expressing high levels of P-Stat5 in Jak2 V617F mice, which the addition of Srsf2 P95H tend to reduce, suggesting an altered Mpl/Jak2 signaling pathway. To validate the hypothesis that Srsf2 P95H negatively interfere with Jak2-mediated signaling in MK, we injected high dose of the thrombopoietin-mimetic romiplostim in mice transplanted with wild-type or Srsf2 P95H BM. Both thrombocytosis and myelofibrosis were significantly reduced in Srsf2 P95H transplanted animals. To further decipher the mechanism by which Srsf2 P95H could alter cell signaling, we performed bulk RNA sequencing on sorted MK. Pathway analysis using gene set enrichment analysis identified mostly a down-regulation of signaling pathways, including JAK/STAT signaling, in Jak2 V617F/Srsf2 P95H compared to Jak2 V617F single mutant cells. Further analysis of splicing events in Srsf2 P95H mutant cells identified an increased exon 14 skipping in Jak2, which was validated by RT-qPCR. Summary: Contrary to EZH2 mutation that promotes JAK2 V617F-induced myelofibrosis in mouse models, heterozygous Srsf2 P95H delays myelofibrosis development in Jak2 V617F-transgenic mice. Srsf2 P95H co-mutation prevents the clonal exhaustion induced by serial transplantation of JAK2 V617F BM cells. This effect is associated with a reduced signaling in MK, which may involve abnormal splicing of signaling components including Jak2 exon 14 skipping. Disclosures Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

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MPL SV, a Unique MPN MPL Splice Variant, Provokes a Fulminant Myeloid Leukemia in a JAK2 V617F Transgenic Mouse Model of Polycythemia Vera

Blood ◽

10.1182/blood.v126.23.484.484 ◽

2015 ◽

Vol 126 (23) ◽

pp. 484-484 ◽

Cited By ~ 1

Author(s):

Jerry L. Spivak ◽

Donna Marie Williams ◽

Zhizhuang Joe Zhao ◽

Ophelia Rogers ◽

Amy S. Duffield ◽

...

Keyword(s):

Platelet Count ◽

Polycythemia Vera ◽

Board Of Directors ◽

Splice Variant ◽

Copy Number ◽

Jak2 V617f ◽

Common Denominator ◽

Wild Type ◽

Advisory Committees ◽

Copy Numbers

Abstract Introduction: The MPN, polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF) are clonal stem cell disorders, which share mutations constitutively activating the physiologic signal transduction pathways for hematopoiesis. Although the MPN have different natural histories, they share in common transformation to myelofibrosis and acute leukemia at differing frequencies and not explainable by a specific mutation. Impaired MPL expression, due to incomplete glycosylation is another common denominator amongst the MPN. Significantly, MPL is the only hematopoietic growth factor receptor expressed in MPN HSC, and we have demonstrated that the PV phenotype in a JAK2 V617F transgenic (V617tg) mouse could be abrogated by elimination of MPL or its ligand, TPO. Impaired MPL expression in the MPN cannot be completely explained by receptor-activated down regulation because not all MPN driver mutations directly activate MPL. However, we have discovered another common denominator in the MPN, variant MPL splicing, eliminating 7 amino acids in the MPL N-terminal domain, a common hotspot for both MPL driver and inactivating mutations, which impairs MPL glycosylation and expression. Methods: To determine the role of the MPL splice variant (MPL SV) in MPN pathophysiology, we cloned the full length MPLSV cDNA and created a transgenic mouse (MPLSV tg) using the same VAV promoter as the V617Ftg mouse to ensure hematopoietic cell-specific transcription. The MPLSV tg mice were produced by pronuclear injection of purified MPLSV cDNA into B6SJLF1 mice. Founders were crossed into a C57Bl/6 background, and then were crossed with V617Ftg mice in either an MPL knockout or wild type background. Mice were phenotyped by blood counts and necropsy with morphologic and immunophenotyping of bone marrow and tumor masses. Results: Amongst 19 founder B6SJLF1 mice, 6 expressed the MPLSV transgene with copy numbers ranging from one to 30; single copy number mice had no hematopoietic phenotype but all mice with higher MPLSV tg copy numbers had thrombocytopenia (median platelet count 500,000/µL; range 200- 600,000; wild type, 750,000/µL; 550-950,000). MPLSV tg mice from several different founders bred into a C57Bl/6 background for 6 generations maintained consistent copy numbers, Mendelian ratios and hematopoietic phenotypes with 100% penetrance and an inverse correlation between MPLSV copy number and the platelet count. The number of double transgenic (V617Ftg/MPLSV tg) offspring observed in the MPLSV tg to V617Ftg crosses were slightly lower than expected (20% vs 25%), indicating reduced embryo viability. Four week old V617Ftg/MPLSV tg mice displayed tumorous abnormalities of the head and hips as well as small size and failure to thrive (median weight 8.5 gms; range 8-9; wild type, 13 gms; 10-14), and enlarged spleens (median weight 0.29 gms; range 0.15-0.64; wild type 0.086 gms; 0.05-0.10). Histologically, the V617Ftg/MPLSV tg-associated head and hip abnormalities represented monomorphic myeloid sarcomas extending through the calvarium both extra and intracranially and from the femur into surrounding muscle. Both the marrow and spleen were diffusely infiltrated by large blasts with abundant basophilic cytoplasm, expressing CD34, CD61 and CD117 but lacking CD127, consistent with a myeloid origin. All V617Ftg/MPLSV tg mice either died or required humane sacrifice by 6 weeks. The extramedullary tumor was transplantable in secondary recipients, and flow cytometry-based phenotyping showed that the tumors (both primary and secondary) were CD34, CD117, CD61 and CD71- positive. Penetrance of the leukemia phenotype was 100% in V617Ftg/MPLSV tg from all founder lines with multiple copies of the MPLSV tg, whether in an MPL knockout or wild type background. The leukemia phenotype was never observed with the MPLSV tg alone or with V617Ftg alone despite observation of over 300 V617Ftg mice up to 50 weeks of age. Conclusion: We identified an MPL SV in human MPN that was functional in mice with a dominant-negative effect with respect to platelet production and at the same time synergized with JAK2 V617F to create a fulminant myeloid malignancy. We have recently shown that knockout of MPL or TPO alone abrogates the PV phenotype in the V617Ftg mouse, whereas the MPLSV uniquely drives a highly penetrant and fulminant leukemia, establishing MPL and TPO as targets for mitigation of malignant transformation in the MPN. Disclosures Spivak: Incyte: Membership on an entity's Board of Directors or advisory committees. Moliterno:incyte: Membership on an entity's Board of Directors or advisory committees.

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Myeloid-Lineage Enhancers Drive Oncogene Synergy and Represent a Novel Therapeutic Target in CSF3R-CEBPA Mutant AML

Blood ◽

10.1182/blood-2019-124773 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 543-543

Author(s):

Theodore Braun ◽

Cody Coblentz ◽

Sarah A Carratt ◽

Mariam Okhovat ◽

Amy Foley ◽

...

Keyword(s):

Clinical Trial ◽

Board Of Directors ◽

Median Survival ◽

Stock Options ◽

Research Funding ◽

Wild Type ◽

Advisory Committees ◽

Dana Farber Cancer Institute ◽

Equity Ownership ◽

Cebpa Mutations

Acute Myeloid Leukemia (AML) results from the stepwise accumulation of mutations from distinct functional classes, ultimately culminating in malignant transformation. Based on their oncogenic activity, mutations can be classified into three distinct groups. Class I mutations activate signaling pathways, produce uncontrolled proliferation, and in isolation produce a myeloproliferative phenotype. Class II mutations result from point mutations or chromosomal translocation events in lineage determining transcription factors, producing differentiation arrest and myelodysplasia in isolation. A classic example of oncogene synergy between distinct mutational classes can be found in the co-occurrence of mutations in the transcription factor CCAAT-enhancer binding protein alpha (CEBPA) with mutations in colony stimulating factor receptor 3 (CSF3R). Mutations in CEBPA occur in approximately 10% of AML where they block differentiation and convey favorable risk. In contrast, CSF3R mutations lead to constitutive receptor activation and uncontrolled neutrophil proliferation. In the absence of co-occurring Class II mutations, membrane proximal CSF3R mutations produce the myeloproliferative neoplasm chronic neutrophilic leukemia (CNL). Interestingly, patients with CEBPA mutant AML that also harbor an oncogenic CSF3R mutation have worse prognosis than those with wild type CSF3R. However, the mechanism underlying this oncogene synergy remains unknown. To model the co-occurrence of these mutations, we expressed CSF3RT618I (The most common membrane proximal CSF3R mutation) in fetal liver hematopoietic stem cells harboring compound heterozygous CEBPA mutations in the endogenous allele (CEBPAK/L). Mice transplanted with mutant CEBPA alone developed a long latency AML with a median survival of 60 weeks. In contrast, mice transplanted with mutant CSF3RT618I/CEBPAK/L cells developed a much more rapid AML with a median survival of 13 weeks. These results were corroborated in an orthogonal model in which mutant CSF3R and a C-terminal mutant CEBPA were retrovirally expressed prior to bone marrow transplant. To dissect the underlying mechanism, we performed a comprehensive transcriptomic and epigenetic analysis on cells expressing each mutation in isolation as well as the combination. This analysis revealed that mutant CSF3R activates a distinct set of enhancers that regulate genes associated with differentiation and drive neutrophil differentiation. Co-expression of mutant CEBPA blocks the activation differentiation-associated enhancers but is permissive to those associated with proliferation. Differentiation but not proliferation-associated enhancers are bound by wild type CEBPA. Thus, the dominant negative impact of mutant CEBPA at these enhancers explains its differential impact on differentiative and proliferative transcriptional programs. Enhancer activation precedes promoter activation and CEBPA mutations are thought to represent early events in AML initiation. The epigenetic mechanism underlying the observed oncogene synergy argues that CEBPA mutations must occur prior to CSF3R to impact differentiation. We therefore developed a retroviral vector system enabling temporal control of Cre-mediated oncogene expression. Using this system, we found that only when mutant CEBPA is expressed prior to mutant CEBPA is differentiation arrest observed. Furthermore, AML develops in vivo only when mutant CEBPA is expressed prior to mutant CSF3R. To develop novel therapeutic strategies for this subclass of AML with adverse prognosis, we performed medium throughput drug screening on CSF3R/CEBPA mutant AML cells and identified sensitivity to inhibitors of JAK/STAT signaling as well as Lysine Demethylase 1 (LSD1). In other subtypes of AML, LSD1 inhibitors activate enhancers associated with differentiation. We confirmed that LSD1 inhibition promotes neutrophilic differentiation in CSF3R/CEBPA and through epigenetic and transcription profiling establish that this occurs via the reactivation of differentiation-associated enhancers. We further found that the combination of ruxolitinib (JAK/STAT inhibitor) and GSK2879552 produce a complete hematologic response and double median survival in mice harboring CSF3R/CEBPA mutant AML. Thus, the combination of JAK/STAT and LSD1 inhibitors represents and exciting therapeutic strategy for CSF3R/CEBPA mutant AML. Disclosures Druker: Celgene: Consultancy; Gilead Sciences: Other: former member of Scientific Advisory Board; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Beat AML LLC: Other: Service on joint steering committee; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Cepheid: Consultancy, Honoraria; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; CureOne: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding.

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Single-Cell Multi-Omics Reveals the Genetic, Cellular and Molecular Landscape of TP53 Mutated Leukemic Transformation in MPN

Blood ◽

10.1182/blood-2021-150191 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3-3

Author(s):

Alba Rodriguez-Meira ◽

Haseeb Rahman ◽

Ruggiero Norfo ◽

Wei Wen ◽

Agathe Chédeville ◽

...

Keyword(s):

Stem Cell ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Clonal Evolution ◽

Wild Type ◽

Advisory Committees ◽

Tp53 Mutations ◽

Molecular Landscape ◽

Mutant Cells

Abstract In myeloid malignancies, presence of 'multi-hit' TP53 mutations is associated with lack of response to conventional therapy and dismal outcomes, particularly when found in combination with a complex karyotype. Therefore, it is crucial to understand the biological basis of TP53-mutant driven clonal evolution, suppression of antecedent clones and eventual disease transformation to inform the development of more effective therapies. Myeloproliferative neoplasms (MPN) represent an ideal tractable disease model to study this process, as progression to secondary acute myeloid leukemia (sAML) frequently occurs through the acquisition of TP53 missense mutations. To characterize tumor phylogenies, cellular hierarchies and molecular features of TP53-driven transformation, we performed single-cell multi-omic TARGET-seq analysis (PMID: 33377019 & 30765193) of 22116 hematopoietic stem and progenitor cells (HSPCs) from 35 donors and 40 timepoints (controls, MPN in chronic phase, pre-AML and TP53-mutated sAML; Figure1a). TARGET-seq uniquely enables single-cell mutation analysis with allelic resolution with parallel transcriptomic and cell-surface proteomic readouts. We invariably identified convergent clonal evolution leading to complete loss of TP53 wild-type alleles upon transformation, including parallel evolution of separate TP53 "multi-hit" subclones in the same patient (n=4/14) and JAK2-negative progression (n=2/14). Complex clonal evolution driven by chromosomal abnormalities (CAs) was present in all patients and TP53 multi-hit HSPCs without CAs were rarely observed. Subclones with recurrent CA such as monosomy 7 showed upregulation of RAS-associated transcription and preferentially expanded in xenograft models. Together, these data indicate that TP53 missense mutation, loss of TP53 wild-type allele and cytogenetic evolution are collectively required for leukemic stem cell (LSC) expansion. Integrated transcriptomic analysis of sAML samples (Figure1b) revealed three major populations: (1) a TP53-mutant cluster (Figure1c) characterized by an erythroid signature (e.g. KLF1, GATA1, GYPA; an unexpected finding as no cases showed diagnostic features of erythroid leukemia), (2) an LSC TP53-mutant cluster (Figure1d) and (3) a TP53-WT preleukemic cluster (Figure1e). The LSC cluster showed dysregulation of key stem cell regulators, from which we derived a novel 48-gene LSC score with prognostic impact in an independent AML cohort (HR=3.13; Figure1f). Importantly, this score was predictive of outcome irrespective of TP53 status for both de novo and sAML, demonstrating its broader potential clinical utility. TARGET-seq analysis uniquely allowed us to characterize rare TP53-WT preleukemic cells (preLSCs), which were almost exclusively confined to the immunophenotypic lineage-CD34+CD38-CD90+CD45RA- HSC compartment. PreLSC from sAML samples presented increased stemness, increased quiescence, aberrant inflammatory signaling and differentiation defects (Figure1g) as compared to HSCs from normal or MPN donors, both at the transcriptional and functional levels through in vitro long-term and short-term cultures. This indicates cell-extrinsic suppression of residual TP53-WT hematopoiesis. Longitudinal analysis of TP53-heterozygous mutant HSPCs at different stages of disease evolution (Figure1a) revealed that aberrant inflammatory signalling (e.g. BST2, IFITM1, IFITM3) in the genetic ancestors of TP53 "multi-hit" LSCs, but not the presence of TP53-mutations alone, was predictive of subsequent transformation. In a mouse model system, TP53-mutant cells challenged with sustained inflammatory stimuli acquired a mean 3-fold competitive advantage in WT: TP53 R172H/+chimeras. This indicates that pro-inflammatory cues from the tumour microenvironment promote fitness advantage of TP53-mutant cells whilst supressing antecedent clones. In summary, we present a comprehensive single-cell multi-omic analysis of the genetic, cellular and molecular landscape of TP53-mediated transformation, providing unique insights into the evolution of chronic hematological malignancies towards an aggressive acute leukemia (Figure1h). Since TP53 is the most commonly mutated gene in human cancer, we anticipate these findings will be of broader relevance to many other cancer types. Figure 1 Figure 1. Disclosures Kretzschmar: Vanadis Diagnostics, a PerkinElmer company.: Current Employment. Drummond: BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Harrison: Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; Constellation Pharmaceuticals: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mead: Abbvie: Consultancy, Honoraria; Celgene/BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Speakers Bureau.

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Bleeding Complications in Patients with Cancer and COVID 19- Analysis from the COVID 19and Cancer Consortium (CCC19) Registry

Blood ◽

10.1182/blood-2021-154314 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4997-4997

Author(s):

Surbhi Shah ◽

Shuchi Gulati ◽

Ang Li ◽

Julie Fu ◽

Vaibhav Kumar ◽

...

Keyword(s):

Mechanical Ventilation ◽

Board Of Directors ◽

Stock Options ◽

Research Funding ◽

Tumor Board ◽

Bleeding Complications ◽

Advisory Committees ◽

Icu Admission ◽

Patients With Cancer ◽

Privately Held

Abstract Background : Patients (pts) with COVID-19 are reported to have increased risk of venous thromboembolism yet bleeding has been an under recognized complication. Rates of bleeding remain unexamined in all patients especially in pts with cancer and COVID-19. Aim: To estimate the incidence of bleeding complication in patients with cancer and COVID 19 Methods: The CCC19 international registry (NCT04354701) aims to investigate complications of COVID-19 in pts with cancer. Our aim was to investigate the frequency of bleeding in hospitalized adult pts with cancer andCOVID-19, enrolled between March 16, 2020 and Feb 8, 2021. The incidence of bleeding complications was captured as defined by CCC19 and included both major and non major bleeding . Associated baseline clinic-pathologic prognostic factors and outcomes such as need for mechanical ventilation, intensive care unit (ICU) admission and mortality rates were assessed Results :3849 pts met analysis inclusion criteria. Bleeding was reported in 276 (7%) pts with median age of 70years; incidence was 6.6 % in females and 7.6 % in males, 6.5% in non-Hispanic white pts, 8.2 % in non-Hispanic Black pts, and 7.8 % in Hispanic pts. 74% had solid cancer and 29% had hematologic malignancies, 33% had received anti-cancer therapy in preceding 30 days, and 8% had surgery within 4weeks. In pts taking antiplatelet or anticoagulant medications at baseline, 7.2% developed bleeding. Need for mechanical ventilation, ICU admission, 30-day mortality, and total mortality were significantly higher in those with bleeding complications compared to those without, p<0.05 Conclusion : We describe the incidence of bleeding in a large cohort of pts with cancer and COVID-19. Bleeding events were observed in those with adverse outcomes including mechanical ventilation, ICU admission, and high mortality; the overall mortality of 43% in patients with bleeding complications is especially notable. This important complication may reflect underlying COVID-19 pathophysiology as well as iatrogenic causes. Figure 1 Figure 1. Disclosures Kumar: Diagnostica Stago: Honoraria. Zon: AMAGMA AND RLZ: Consultancy, Current holder of individual stocks in a privately-held company. Byeff: Pfizer, BMS, Takeda,Teva, Merck, United health: Consultancy, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Nagaraj: Novartis: Research Funding. Hwang: astrazaneca,Merck,bayer, Genentech: Consultancy, Research Funding. McKay: Myovant: Consultancy; Bayer: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Calithera: Membership on an entity's Board of Directors or advisory committees; Tempus: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tempus: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Dendreon: Consultancy; Caris: Other: Serves as a molecular tumor board ; Vividion: Consultancy; Sorrento Therapeutics: Consultancy; Bayer: Research Funding. Warner: Westat, Hemonc.org: Consultancy, Current holder of stock options in a privately-held company. Connors: Pfizer: Honoraria; CSL Behring: Research Funding; Alnylam: Consultancy; Bristol-Myers Squibb: Honoraria; takeda: Honoraria; Abbott: Consultancy. Rosovsky: Janssen: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Inari: Consultancy, Membership on an entity's Board of Directors or advisory committees; Dova: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Enhancement of Fluoroquinolone Activity by C-8 Halogen and Methoxy Moieties: Action against a Gyrase Resistance Mutant of Mycobacterium smegmatis and a Gyrase-Topoisomerase IV Double Mutant of Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.10.2703-2709.2001 ◽

2001 ◽

Vol 45 (10) ◽

pp. 2703-2709 ◽

Cited By ~ 34

Author(s):

Tao Lu ◽

Xilin Zhao ◽

Xinying Li ◽

Alex Drlica-Wagner ◽

Jian-Ying Wang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Mycobacterium Smegmatis ◽

Double Mutant ◽

Structure Refinement ◽

Fluoroquinolone Resistance ◽

Wild Type ◽

Topoisomerase Iv ◽

Methoxy Groups ◽

Bactericidal Activities ◽

Mutant Cells

ABSTRACT The increasing prevalence of antibiotic resistance among bacterial pathogens prompted a microbiological study of fluoroquinolone structure-activity relationships with resistant mutants. Bacteriostatic and bactericidal activities for 12 fluoroquinolones were examined with a gyrase mutant of Mycobacterium smegmatis and a gyrase-topoisomerase IV double mutant of Staphylococcus aureus. For both organisms C-8 halogen and C-8 methoxy groups enhanced activity. The MIC at which 99% of the isolates tested were inhibited (MIC99) was reduced three- to fivefold for the M. smegmatis mutant and seven- to eightfold for theS. aureus mutant by C-8 bromine, chlorine, and methoxy groups. With both organisms a smaller reduction in the MIC99 (two- to threefold) was associated with a C-8 fluorine moiety. In most comparisons with M. smegmatis the response to a C-8 substituent was similar (within twofold) for wild-type and mutant cells. In contrast, mutant S. aureuswas affected more than the wild type by the addition of a C-8 substituent. C-8 halogen and methoxy groups also improved the ability to kill the two mutants and the respective wild-type cells when measured with various fluoroquinolone concentrations during an incubation period equivalent to four to five doubling times. Collectively these data help define a group of fluoroquinolones that can serve (i) as a base for structure refinement and (ii) as test compounds for slowing the development of fluoroquinolone resistance during infection of vertebrate hosts.

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Induced Resistance to Bortezomib in Preclinical Model of Waldenstrom Macroglobulinemia Is Associated with Bcl-2 Upregulation.

Blood ◽

10.1182/blood.v114.22.4919.4919 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4919-4919 ◽

Cited By ~ 1

Author(s):

Kasyapa Chitta ◽

Jean-Gabriel Coignet ◽

Sakina Sojar ◽

Pushpankur Ghoshal ◽

Kiersten M. Miles ◽

...

Keyword(s):

Multidrug Resistance ◽

Cell Line ◽

Board Of Directors ◽

Induced Resistance ◽

Wild Type Cell ◽

Preclinical Model ◽

Wild Type ◽

Advisory Committees ◽

Proteasomal Activity ◽

Resistant Cells

Abstract Abstract 4919 Waldenstrom's macroglobulinemia (WM) is characterized by the presence of lymphoplasmacytic cells in the bone marrow and the secretion of IgM monoclonal antibody in the serum. Several conventional therapies are available but the disease remains incurable. Recently, bortezomib (a proteasomal inhibitor) has shown promising anti-WM activity with enhanced responses when combined with traditional therapies. Resistance to bortezomib therapy is an important event that is associated with continued treatment. In order to understand the mechanism of bortezomib resistance in WM we exposed BCWM.1 (a known WM cell line) in vitro to increasing concentrations of bortezomib over prolonged periods of time and isolated the bortezomib resistant clone (BCWM.1/BR). This clone was compared with its parent wild type cell line (BCWM.1/WT). Investigation to understand the susceptibility of BCWM.1/Br cells to various therapeutic agents showed that these cells are resistant to many of the agents such as melaphalan, fludarabine or doxorubicin. Interestingly, verapamil, a broad spectrum inhibitor of multidrug resistance, failed to reverse the resistance induced by bortezomib indicating that bortezomib resistance is not because of an activation of multidrug resistance in these cells. While BCWM.1/WT cells showed an IC50 of 7.8nM when treated for 72h with bortezomib, the BCWM.1/BR cells were not inhibited at any concentration of the compound up to 100nM. Furthermore, the cells with the acquired resistance showed a 4 fold increase in the proteasomal activity as measured by the release of a fluorescent product (7-Amino-4-methylcoumarin (AMC)) from its peptide substrate, suc-LLVY-AMC. Biochemical analysis further revealed that many of the proteasomal components are altered in BCWM.1/BR cells as compared to their parental control cells. Interestingly, protein levels of two of the proteasomal catalytic subunits, PSMB5 and PSMB9 are upregulated in resistant cells suggesting a reason for the enhanced proteasomal activity of these cells. The resistant cells showed an altered gene expression profile that indicates a transformation of the parental wild type cell line to acquire resistance. A comparative analysis of the signal transduction pathways operated in these cells showed that many of the activation and cell survival pathways that are present in BCWM.1 cells are inhibited in the resistant cells. For example, BCWM.1 cells show a constitutive activation of AKT and ERK1/2 which are inhibited in the resistant cells thus making them insensitive to the inhibitors of these pathways. Similarly, HSP27 which was earlier shown to contribute to bortezomib induced resistance was completely inhibited in BCWM.1 resistant cells. Interestingly, there is an increase in Bcl-2 protein in BCWM.1/BR cells as compared to WT cells indicating that the resistant cells might be dependent on Bcl-2 family for their survival. Inhibition of Bcl-2 induced potent apoptosis in BCWM.1/BR cells. Thus the results presented here indicate that acquired bortezomib resistance in BCWM.1 cells alters their proteasomal activity, cellular signaling pathways to make them resistant to many of the known therapies but these cells retain the Bcl-2 mediated pathway for targeting thus inhibitors of Bcl-2 may be used in therapy against bortezomib-resistant WM. Disclosures Chanan-Khan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immunogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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In Vivo Genome Editing of Liver Albumin for Therapeutic Gene Expression: Rescue of Hemophilic Mice Via Integration of Factor 9

Blood ◽

10.1182/blood.v120.21.751.751 ◽

2012 ◽

Vol 120 (21) ◽

pp. 751-751 ◽

Cited By ~ 1

Author(s):

Xavier M Anguela ◽

Rajiv Sharma ◽

Yannick Doyon ◽

Sunnie Y Wong ◽

David E Paschon ◽

...

Keyword(s):

Board Of Directors ◽

Transgene Expression ◽

Factor Ix ◽

Safe Harbor ◽

Wild Type ◽

Advisory Committees ◽

Aav Vector ◽

Children's Hospital ◽

Children’S Hospital

Abstract Abstract 751 Gene correction using zinc finger nuclease (ZFN) technology can be applied to target virtually any locus in the human genome. Beyond correcting mutated genes causative of disease, ZFNs can also be utilized to target transgene insertion into genomic “safe harbors.” Ideally, specific gene targeting to such “safe harbor” sites would (i) ensure therapeutically relevant levels of transgene expression and (ii) tolerate transgene addition without deleterious effect on the host organism. For liver-derived protein replacement, albumin represents an attractive target locus. Firstly, albumin is very highly expressed exclusively in the liver, thus targeting of a relatively small percentage of alleles should yield therapeutically relevant levels of liver-specific transgene expression. Second, the reduction or complete absence of albumin in animals and even humans (analbuminemia) produces surprisingly few symptoms. Here, we sought to investigate whether ZFN-mediated targeted insertion of a promoter-less copy of the human F9 cDNA at the mouse albumin locus could result in human Factor IX production and successfully correct the hemophilic phenotype in mice. To address this question, we constructed an AAV vector encoding a pair of ZFNs targeting intron 1 of the mouse albumin locus (AAV8-mAlb-ZFN) and a donor AAV vector (AAV8-Donor) harboring a partial cDNA cassette containing exons 2–8 of the wild-type human F9 gene flanked by sequences lacking significant homology to the mouse genome. Co-delivery of 1e11 vg of AAV8-mAlb-ZFN along with 5e11vg of AAV8-Donor resulted in stable (>12wk) circulating F.IX levels of 1600–3200 ng/mL (32–64% of normal). As a control, mice injected with the AAV8-Donor along with an AAV vector encoding a ZFN pair targeting an unrelated locus exhibited background F.IX levels (∼50 ng/mL). A dose-response study was performed by administering a fixed dose of donor (5e11 vg/mouse) with decreasing doses of AAV8-mAlb-ZFN (1e11, 1e10 and 1e9 vg/mouse). Human F.IX levels increased as a function of ZFN dose in the range tested (3260±480, 225±43 and 31±4 ng/mL at the high, medium and low dose, respectively). Importantly, these results showed that donor homology to the target site is not required to achieve robust levels of gene addition to the albumin locus in adult mice, thus permitting the design of donor vectors harboring corrective copies of transgenes up to the maximum AAV packaging capacity of ∼4.7 Kb. Albumin and factor IX are both synthesized as pre-propeptides and turned into propeptides after the signal peptide is removed. Expression of human F9 exons 2–8 spliced with mouse albumin exon 1 is expected to yield a chimeric propeptide. The first 2 N-terminal amino acids would originate from proalbumin, followed by a Val to Leu mutation at position −17 of the hF.IX propeptide and 16 aa encoded by human F9. To evaluate whether this chimeric human F.IX derived from gene addition to the albumin locus would be processed correctly and normalize the prolonged clotting times in hemophilia B (HB) mice, we injected 1e11 vg of AAV8-mAlb-ZFN and 5e11vg of AAV8-Donor into HB animals. Two weeks post-treatment, hF.IX antigen levels were in the range of 20% of normal and activated partial thromboplastin time, a measurement of clot formation, was corrected to wild-type levels (42 seconds), from an average of 70 seconds pre-treatment. Thus expression of a therapeutic protein (F.IX) from the albumin locus is shown to correct the HB disease phenotype in vivo. In summary, these data provide the first demonstration of ZFN-mediated in vivo genome editing of a safe harbor locus for therapeutic protein production. While we provide here a proof of principle establishing phenotypic correction of hemophilia B, appropriately designed donors could expand this strategy. Most importantly the magnitude of albumin expression (>15 g / day) should enable production of a diverse range of transgenes at therapeutically consequential levels. Disclosures: Anguela: The Children's Hospital of Philadelphia: Patents & Royalties. Sharma:The Children's Hospital of Philadelphia: Patents & Royalties. Doyon:Sangamo BioSciences, Inc.: Employment. Wong:Sangamo BioSciences, Inc.: Employment. Paschon:Sangamo BioSciences, Inc.: Employment. Gregory:Sangamo BioSciences, Inc.: Employment. Holmes:Sangamo BioSciences, Inc.: Employment. Rebar:Sangamo BioSciences, Inc.: Employment. High:Shire Pharmaceuticals: Consultancy; Sangamo Biosciences, Inc: Collaborator, Collaborator Other; Novo Nordisk: Visiting Professor, Visiting Professor Other; Genzyme, Inc: Membership on an entity's Board of Directors or advisory committees; The Children's Hospital of Philadelphia: Patents & Royalties; Bluebird Bio, Inc: Membership on an entity's Board of Directors or advisory committees.

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Ponatinib In Heavily Pretreated Patients With Chronic Phase Chronic Myeloid Leukemia (CP-CML): Management Of Adverse Events (AEs)

Blood ◽

10.1182/blood.v122.21.1496.1496 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1496-1496 ◽

Cited By ~ 3

Author(s):

Philipp D. Le Coutre ◽

Dong-Wook Kim ◽

Javier Pinilla-Ibarz ◽

Ronald Paquette ◽

Charles Chuah ◽

...

Keyword(s):

Board Of Directors ◽

Stock Options ◽

Research Funding ◽

Peripheral Vascular ◽

Vascular Events ◽

Advisory Committees ◽

Dose Modification ◽

Advisory Boards ◽

Initial Onset ◽

Grade 3

Abstract Background Ponatinib is a potent oral pan–BCR-ABL tyrosine kinase inhibitor (TKI) with activity against native and mutant BCR-ABL. The efficacy and safety of ponatinib (45 mg once daily) in pts with CP-CML were evaluated in the phase 2 PACE trial. Objectives To review the management of treatment-related AEs (TRAEs) that emerged during therapy with ponatinib in the PACE trial. Methods A total of 270 CP-CML pts (267 in efficacy population) resistant or intolerant to dasatinib or nilotinib or with the T315I mutation were enrolled in this ongoing, phase 2, international, open-label clinical trial. The primary endpoint was major cytogenetic response (MCyR) at any time within 12 mos. Safety monitoring included collection of AEs, and the following variables were evaluated: incidence, severity, time to onset, duration, and management. Select TRAEs are discussed. Data as of 01 Apr 2013 are reported, with a median follow-up of 20 (0.1–28) mos. Minimum follow-up for pts remaining on study was 18 mos. Results Median age was 60 (18-94) yrs; median time from diagnosis to first dose was 7 (0.5-27) yrs; 93% had ≥2 prior TKIs, 60% ≥3. Ponatinib demonstrated significant activity in CP-CML pts: 56% MCyR, 46% CCyR, and 36% MMR. At the time of analysis, 60% of pts remained on study. The most frequent reasons for discontinuation were AEs (14%) and progression (8%). The most common hematologic TRAE was thrombocytopenia (41% any grade, 32% grade 3/4). The incidence by time to initial onset is shown below (Figure). Pts experienced thrombocytopenia for a median total duration of 166 days (64% of whom had >1 event) and typically required dose modification: 13% drug withdrawn, 40% dose reduced, 29% dose interrupted only, 17% no dose modification. Among pts with thrombocytopenia, 27% required a platelet transfusion. Thirteen percent of CP-CML pts experienced treatment-related neutropenia and thrombocytopenia. The most common nonhematologic TRAE was rash (39% any grade, 4% grade 3/4), which includes erythematous, macular, and papular rash. Pts experienced rash for a median total duration of 65 days (46% of whom had >1 event) and most did not require dose modification: 0% drug withdrawn, 15% dose reduced, 11% dose interrupted only, 73% no dose modification. One additional pt discontinued due to grade 2 treatment-related exfoliative rash. Pancreatitis was observed (7% any grade, 6% grade 3/4). Median duration was 5 days. Pts were typically managed with dose modification: 5% drug withdrawn, 58% dose reduced, 32% dose interrupted only, 5% no dose modification. Treatment-emergent cardiovascular events were observed in 8% of pts and treatment-emergent cerebrovascular or peripheral vascular events in 11%. Cardiovascular events were considered treatment-related in 4%; cerebrovascular or peripheral vascular events were also considered treatment-related in 4%. The median time to initial onset was 9 mos for cardiovascular and 11 mos for cerebrovascular or peripheral vascular events. The median duration was 6 and 97 days, respectively. Management of pts with cardiovascular AEs: 20% drug withdrawn, 10% dose reduced, 40% dose interrupted only, 30% no dose modification. Management of pts with cerebrovascular or peripheral vascular AEs: 8% drug withdrawn; 8% dose reduced; 17% dose interrupted only; 67% no dose modification. Conclusions Ponatinib has robust antileukemic activity in heavily pretreated CP-CML pts (93% of whom received ≥2 prior TKIs). Treatment-related thrombocytopenia and pancreatitis generally occurred early in therapy and were manageable with dose modification. Treatment-related rash generally occurred early in therapy, was mild-to-moderate in severity, managed without the need for dose modification, and rarely led to discontinuation. Management of treatment-related arterial thrombotic events varied; pts with predisposing cardiovascular risk factors should be monitored closely and managed accordingly. ClinicalTrials.gov ID: NCT01207440 aIncidence = (number of pts with initial onset during time interval) / (number of pts dosed during time interval [N] excluding those who previously experienced the event) X 100 Disclosures: Le Coutre: Novartis: Research Funding; Novartis, BMS, Pfizer: Honoraria. Kim:BMS, Novartis, IL-Yang: Consultancy; BMS, Novartis, Pfizer, ARIAD, IL-Yang: Research Funding; BMS, Novartis, Pfizer, IL-Yang: Honoraria; BMS, Novartis, Pfizer: Speakers Bureau; BMS, Pfizer: Membership on an entity’s Board of Directors or advisory committees. Pinilla-Ibarz:Novartis, Ariad: Research Funding; Novartis, Ariad, BMS and Pfizer: Speakers Bureau. Paquette:Ariad, BMS, Novartis: Consultancy; Ariad, BMS, Novartis: Honoraria; Ariad, BMS, Novartis: Speakers Bureau. Chuah:Novartis, BMS: Honoraria. Nicolini:Novartis, ARIAD, Teva: Consultancy; Novartis, BMS: Research Funding; Novartis, BMS, Teva, Pfizer, ARIAD: Honoraria; Novartis, BMS, TEva: Speakers Bureau; Novartis, ARIAD, Teva, Pfizer: Membership on an entity’s Board of Directors or advisory committees. Apperley:Novartis: Research Funding; Ariad, Bristol Myers Squibb, Novartis, Pfizer, Teva: Honoraria. Talpaz:Ariad, BMS, Sanofi, INCYTE: Research Funding; Ariad, Novartis: Speakers Bureau; Ariad, Sanofi, Novartis: Membership on an entity’s Board of Directors or advisory committees. DeAngelo:Ariad, Novartis, BMS: Consultancy. Abruzzese:BMS, Novartis: Consultancy. Rea:BMS, Novartis, Pfizer, Ariad, Teva: Honoraria. Baccarani:ARIAD, Novartis, BMS: Consultancy; ARIAD, Novartis, BMS, Pfizer, Teva: Honoraria; ARIAD, Novartis, BMS, Pfizer, Teva: Speakers Bureau. Muller:Novartis, BMS, ARIAD: Consultancy; Novartis, BMS: Research Funding; Novartis, BMS, ARIAD: Honoraria. Gambacorti-Passerini:Pfizer: Research Funding; Pfizer, BMS: Honoraria. Lustgarten:ARIAD: employees of and own stock/stock options in ARIAD Pharmaceuticals, Inc Other, Employment. Yanase:ARIAD: employees of and own stock/stock options in ARIAD Pharmaceuticals, Inc. Other, Employment. Turner:ARIAD: Employment. Haluska:ARIAD: employees of and own stock/stock options in ARIAD Pharmaceuticals, Inc Other, Employment. Deininger:BMS, ARIAD, NOVARTIS: Consultancy; BMS, NOVARTIS, CELGENE, GILEAD: Research Funding; ARIAD, NOVARTIS: Advisory Boards, Advisory Boards Other. Hochhaus:Ariad, Novartis, BMS, MSD, Pfizer: Research Funding; Novartis, BMS, Pfizer: Honoraria. Hughes:Novartis, BMS, ARIAD: Honoraria, Research Funding. Goldman:ARIAD: Honoraria. Shah:Ariad, Bristol-Myers Squibb: Consultancy, Research Funding. Kantarjian:ARIAD, Novartis, BMS, Phizer: Research Funding. Cortes:Ariad, Pfizer, Teva: Consultancy; Ariad, BMS, Novartis, Pfizer, Teva: Research Funding.

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The CSF3R T618I Mutation Found In Chronic Neutrophilic Leukemia Removes An O-Linked Glycosylation Site and Increases Receptor Dimerization

Blood ◽

10.1182/blood.v122.21.270.270 ◽

2013 ◽

Vol 122 (21) ◽

pp. 270-270

Author(s):

Julia E. Maxson ◽

Jason Gotlib ◽

Daniel A. Pollyea ◽

Angela G. Fleischman ◽

Anupriya Agarwal ◽

...

Keyword(s):

Clinical Trials ◽

Board Of Directors ◽

Research Funding ◽

Conflicts Of Interest ◽

Glycosylation Site ◽

Wild Type ◽

Advisory Committees ◽

Advisory Boards ◽

Financial Interest ◽

Truncation Mutant

Abstract Background We have recently identified mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) in ∼60% of chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) patients (Maxson et al, NEJM 2013). These mutations fall into two categories: membrane proximal point mutations (the most common of which is T618I) and truncation mutations. Drug and siRNA screening of primary patient samples revealed that the two classes of CSF3R mutations exhibit differential sensitivity to inhibition of SRC or JAK kinases. CSF3R truncation mutations conferred sensitivity to SRC family kinase inhibition, while CSF3R membrane proximal mutations (T618I) conferred sensitivity to JAK kinase inhibition. A patient with the T618I membrane proximal mutation responded to treatment with the FDA approved JAK inhibitor, ruxolitinib. CSF3R truncation mutations have also been observed in a subset of severe congenital neutropenia patients who are at high risk for development of acute myeloid leukemia. Prior studies in this context have shown that truncation mutations result in loss of endocytic and degradation motifs, leading to increased expression of the receptor. The differences in signaling and drug sensitivity of these mutation classes suggest that membrane proximal mutations may activate CSF3R signaling through a distinct, as-yet unknown mechanism. Furthermore, a subset of CNL patients harbor both membrane proximal and truncation mutations on the same allele, though the consequences of these compound mutations are not yet known. Methods CSF3R expression level and banding pattern were assessed by immunoblot of lysates from 293T17 cells transfected with wild type, membrane proximal mutant, or truncation mutant CSF3R. O-linked glycosylation was removed from the receptor by treatment with O-glycosidase and neuraminidase. Ligand independence of the CSF3R mutants was analyzed in murine interleukin-3 (IL3)-dependent Ba/F3 cells. CSF3R dimerization was assessed by co-transfecting CSF3R-Flag and CSF3R-V5 tagged constructs and then immunoprecipitating CSF3R-Flag and detecting co-immunoprecipitation of the CSF3R-V5 by immunoblot. Transforming potential of the CSF3R compound mutations relative to the corresponding point or truncation mutations was assessed by analyzing IL3-independent growth of Ba/F3 cells or mouse bone marrow colony formation. Results To better understand the functional and biochemical differences between membrane proximal and truncation mutant CSF3R, we examined transformation potential, requirement for ligand, and expression patterns in Ba/F3 and 293T17 cells. We found membrane proximal mutations to exhibit rapid transformation potential and ligand independence, while truncation mutations exhibited delayed transformation and ligand hypersensitivity. Unlike the truncation mutations, which induce dramatic overexpression of CSF3R, the T618I mutation did not result in overexpression of the receptor but instead induced a shifted banding pattern, indicative of altered protein modification. We examined the amino acid sequence surrounding the membrane proximal mutations and found residue T618 to be part of a consensus motif for O-glycosylation, wherein wild type CSF3R is O-glycosylated and the T618I mutation abrogates this O-glycosylation event. Furthermore, the T618I mutation exhibited increased receptor dimerization compared to wild type CSF3R, which likely explains its ligand independence. Finally, we found that CSF3R compound mutations have increased transforming potential in Ba/F3 and murine bone marrow colony assays compared with either class of single mutation, further underscoring the different mechanisms of action of the membrane proximal and truncation mutations. Conclusion CSF3R represents a promising therapeutic target for patients with CNL. We show that T618I, the most common CSF3R mutation in CNL, is part of an O-linked glycosylation site. Mutation of this residue leads to loss of O-linked glycosylation and represents a novel mechanism of homodimeric cytokine receptor activation. CSF3R compound mutations are more rapidly transforming relative to the membrane proximal or truncation mutations alone, warranting their further investigation for patient prognosis and therapy. Disclosures: Off Label Use: Ruxolitinib - a JAK1/2 inhibitor that we propose can be used off-label for disease management of CSF3R-mutant neutrophilic leukemia. Gotlib:Incyte: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Travel Support Other. Fleischman:Incyte: Speakers Bureau. Collins:Genoptix: Membership on an entity’s Board of Directors or advisory committees. Oh:Incyte Corporation: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau. Deininger:Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Bristol-Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding. Druker:Bristol-Myers Squibb: PI or co-investigator on BMS clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Novartis: PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Incyte: PI or co-investigator on clinical trials., PI or co-investigator on clinical trials. Other. Tyner:Incyte Corporation: Research Funding.

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Role Of B Cell Tolerance In PF4/Heparin Antibody Production

Blood ◽

10.1182/blood.v122.21.2396.2396 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2396-2396

Author(s):

Yongwei Zheng ◽

Alexander W Wang ◽

Mei Yu ◽

Anand Padmanabhan ◽

Benjamin E Tourdot ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Board Of Directors ◽

Cell Tolerance ◽

Inflammatory Stimulus ◽

Wild Type ◽

B Cell Tolerance ◽

Advisory Committees

Abstract Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of heparin, platelet factor 4 (PF4) and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, heparin, a glycosoaminoglycan, and PF4 are normal body constituents and it is as yet unclear what triggers the initial induction of pathogenic antibodies. Here we described detection of B cells among peripheral blood mononuclear cells (PBMCs) from each of 9 healthy adults that produced PF4/heparin-specific IgM antibodies following in vitro stimulation with ubiquitous pro-inflammatory molecules containing unmethylated CpG dinucleotides derived from bacterial and viral DNA. PF4/heparin-specific IgM-generating B cells were present at a frequency of at least 0.03 to 1 per thousand B cells present in the PBMC population. Similarly, splenic B cells isolated from unmanipulated wild-type mice consistently produced PF4/heparin-reactive antibodies following in vitro stimulation with CpG. In addition, wild-type mice produced PF4/heparin-reactive antibodies upon in vivo challenge with CpG whereas unchallenged wild-type mice did not. These findings demonstrate that both humans and mice possess pre-existing, inactive and tolerant PF4/heparin-specific B cells. We suggest that tolerance can be broken by a strong inflammatory stimulus, leading to activation of these B cells and production of antibodies that recognize PF4/heparin in vitro and in vivo. Consistent with this concept, mice lacking protein kinase Cd (PKCd), a signaling molecule of the B-cell survival factor BAFF (B-cell activation factor), that are known to have breakdown of B-cell tolerance to self-antigens, spontaneously produced anti-PF4/heparin antibodies in the absence of an inflammatory stimulus. Taken together, these findings demonstrate that breakdown of tolerance can lead to PF4/heparin-specific antibody production and that B-cell tolerance plays an important role in HIT pathogenesis. Disclosures: White II: Bayer: Membership on an entity’s Board of Directors or advisory committees; CSL-Behring: Membership on an entity’s Board of Directors or advisory committees; NIH: Membership on an entity’s Board of Directors or advisory committees; Asklepios: Membership on an entity’s Board of Directors or advisory committees; Wyeth: Membership on an entity’s Board of Directors or advisory committees; Entegrion: Membership on an entity’s Board of Directors or advisory committees; Biogen: Membership on an entity’s Board of Directors or advisory committees; Baxter: Membership on an entity’s Board of Directors or advisory committees.

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