Primary Myelofibrosis Patient Research Articles

The telomerase inhibitor imetelstat differentially targets JAK2V617F versus CALR mutant myeloproliferative neoplasm cells and inhibits JAK-STAT signaling.

Imetelstat shows activity in patients with myeloproliferative neoplasms, including primary myelofibrosis (PMF) and essential thrombocythemia. Here, we describe a case of prolonged disease stabilization by imetelstat treatment of a high-risk PMF patient enrolled into the clinical study MYF2001. We confirmed continuous shortening of telomere length (TL) by imetelstat treatment but observed emergence and expansion of a KRAST58I mutated clone during the patient's clinical course. In order to investigate the molecular mechanisms involved in the imetelstat treatment response, we generated induced pluripotent stem cells (iPSC) from this patient. TL of iPSC-derived hematopoietic stem and progenitor cells, which was increased after reprogramming, was reduced upon imetelstat treatment for 14 days. However, while imetelstat reduced clonogenic growth of the patient's primary CD34+ cells, clonogenic growth of iPSC-derived CD34+ cells was not affected, suggesting that TL was not critically short in these cells. Also, the propensity of iPSC differentiation toward megakaryocytes and granulocytes was not altered. Using human TF-1MPL and murine 32DMPL cell lines stably expressing JAK2V617F or CALRdel52, imetelstat-induced reduction of viability was significantly more pronounced in CALRdel52 than in JAK2V617F cells. This was associated with an immediate downregulation of JAK2 phosphorylation and downstream signaling as well as a reduction of hTERT and STAT3 mRNA expression. Hence, our data demonstrate that imetelstat reduces TL and targets JAK/STAT signaling, particularly in CALR-mutated cells. Although the exact patient subpopulation who will benefit most from imetelstat needs to be defined, our data propose that CALR-mutated clones are highly vulnerable.

Complement Factor I (CFI) Deficiency Is Linked to Alterations in Humoral Immunity and Accelerated Development of JAK2V617F-Dependent Phenotype

Background: Inflammation is an important phenomenon in hematologic malignancies, though its drivers are not well defined. Within the hematopoietic system, the complement cascade plays a complex role in regulating hematopoietic stem and progenitor cell (hSPC) homing and exit to systemic circulation (Lenkiewicz et al, Frontiers in Immunology 2019). The role the complement system plays in both normal and malignant hSPC egress from the bone marrow has been recognized, though its role in pathogenesis of hematologic malignancies is not well established. Complement cascade contributions to inflammation-mediated pathologies expanded the traditional view of the cascade from an acute process at the site of injury or pathogen invasion, to a mediator of chronic inflammation (Markiewskiet al, American Journal of Pathology 2007). We previously uncovered a rare single nucleotide polymorphism in an inhibitor of the complement cascade Complement Factor I - CFI (CFIG119R). This loss-of-function mutation was detected using whole genome sequencing in 20% of patients with primary myelofibrosis (PMF). In a separate PMF patient cohort, we observed an increase in global complement activity through elevated CH50 levels and decrease in C3 levels (Sadler et al, Blood 2021). There remains a gap in the understanding of how the complement cascade in general and CFI specifically contribute to development and progression of hematologic malignancies. Therefore, we established a murine model of CFI deficiency to determine mechanistic details of complement cascade contribution to both systemic and local inflammation and development of JAK2V617F-dependent pathologies. Methods: To determine the effect of CFI deficiency on homeostasis of hematopoietic system a CFI KO mouse model was generated by floxing exon 2 of the Cfi gene and removing it using CRISPR/Cas9-driven cre recombination. Inactivation of Cfi was confirmed by genotyping. Frequencies of granulocytes, B cells, plasmacytes, T cells, macrophages/monocytes, platelets, erythroid cells were assessed by FACS within the fraction of bone marrow mononuclear cells in 4-week-old CFIWT, HET (n=8) and KO (n=8) mice using established profiling protocols (Chorzalska et al, Blood 2018). Complete blood counts (CBCs) were performed on peripheral blood of 4-week-old Cfi WT (n=20), HET (n=26) and KO (n=10) mice. To determine the effect of CFI deficiency on JAK2V617F-driven phenotypes we performed colony forming assay using CFI WT (n=4, 2F, 2M)) or HET (n=4, 2F, 2M) bone marrows that were transduced with JAK2 V617F encoding retrovirus. Results: CFI KO and HET animals were viable and fertile. CBC analyses of CFI KO, HET vs. WT 4-week-old mice indicated no significant changes with an exception of a significant increase in red blood cell width distribution (RDW) in KO vs. WT and KO vs. HET peripheral blood samples suggestive of a mild anemia. Trends towards granulocytosis and monocytosis as well as leukopenia, erythropenia and decrease in hemoglobin levels were noted. Changes within the red blood cell compartment were confirmed by FSCS. In addition, flow cytometry data showed a significant decrease in CD19+ B-cells in CFI HET vs. WT animals, suggestive of dysregulation of B-cell maturation. Finally, colony forming unit assays showed 59% increase in number of colonies formed by CFI HET/JAK2 V617F+ vs. CFI WT/JAK2 V617F+ bone marrow cells indicative that loss of one copy of Cfi is sufficient to exacerbate JAK2V617F-dependent cellular phenotype. Conclusions: Early characterization of our newly generated CFI KO mouse model shows a phenotype indicative of CFI-loss induced changes within the homeostasis of hematopoietic system affecting red blood cell and B-cell compartments. Our data also indicate that loss of one copy of Cfi may accelerate cell growth and proliferation characteristics of JAK2 V617F-dependent myeloproliferative neoplasms. Detailed analysis of the CFI KO model may uncover novel therapeutically targetable mechanisms that link the pro-inflammatory and myeloproliferative phenotypes of some stem cell disorders, including JAK2 V617F-dependent MPNs.

Frequency of JAK2 and MPL Mutation in BCR/ABL Negative Myelofibrosis in KPK

Introduction: BCR-ABL1-negative myeloproliferative disorders are a sub-group of myeloproliferative neoplasms (MPNs) that consist of polycythemia Vera (PV), Essential thrombocythemia (ET) and Primary Myelofibrosis (PMF). Over the past decade, the morphological and clinical division of myeloproliferative neoplasms (MPN) has changed to a classification that describes the molecular heterogeneity and is unique to this subgroup of haematological malignancies. This includes alterations in Janus kinase 2 (JAK2), and MPL genes. Objective: To determine the frequency of JAK2 (p.V617F) and MPL (p.W515L) mutation in primary myelofibrosis in KPK province of Pakistan. Materials and Methods: Fifty patients with PMF were enrolled in the study. JAK2 mutation status was determined using allele specific primers and MPL mutation was detected by direct Sanger sequencing technique. The data was analyzed using BioEdit by aligning the sequence data with reference genome hg19 assembly. Results: Among 50 patients, 41 patients were diagnosed with PMF, while 9 patients had secondary myelofibrosis i.e. Post PV-MF and Post ET-MF. Out of the 41 PMF patient 2 patients had MPL gene variation, while one of the Post ET –MF had a MPL gene variation. Forty eight (96%) were positive for JAK2 mutation. Five patients who had MPL mutation also showed JAK 2 mutation. Two of the MPL positive patients were also positive for JAK2 mutation. Conclusion: We reported rate of recurrence of JAK2 mutation in 96% of the cases and MPL exon 10 mutations in 6% of the cases.

Establishment and Characterization of a Human Primary Myelofibrosis Cell Line

Aims: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by clonal proliferation of stem cells and myeloid cells. JAK2, CARL, and MPL mutations could be detected in most patients, but 15% of PMF patients could be absent of these three gene mutations. PMF has shorter median survival time and poorer prognosis when compared with closely related MPNs, polycythemia vera (PV) and essential thrombocythemia (ET). Allogenic hematopoietic stem cells (allo-HSCT) is the only curative therapy for PMF patients, however transplantation-related complications and deaths were observed in more than 50% patients. Hydroxyurea and Ruxolitinib are commonly used drugs to improve clinical symptoms of PMF patients, but lack of anti-tumor activities. These drugs could not reverse bone marrow fibrosis or induce cytogenetic remission. Basic research work of PMF is limited as to the lack of human PMF cell line. Here, we aimed to establish and characterize a patient-derived human PMF cell line, which would provide a useful tool for PMF research and screenings for novel drugs.Methods: The bone marrow cells were obtained from a 61-year-old male patient, who was diagnosed as PMF for 2 years. Mono-nuclear cells were isolated and cultured in a continuous culture system. The cells were characterized by different methods, including STR profiling, morphology observation with Giemsa staining, flow cytometry, chromosome analysis, Epstein-Barr virus (EBV) detection, mycoplasma detection, whole-genome-exon sequencing, and cell proliferation assay.Results: The cells have continued to grow in liquid culture for more than 50 passages using the same culture conditions. Doubling time was about 48-72 hours. The STR profiling identified that the cultured cells and the patient's bone marrow cells were from the same origin. The cells are free from EBV and mycoplasma. Giemsa-Wrights staining showed primitive erythrocytes (Figure A), which was consistent with CD71 positive detected by flow cytometry. Chromosome analysis revealed a hyperdiploid karyotype, which was 47, XY,+12[1]/48,idem,+9[9]. Whole-genome-exon sequencing identified mutations of ASXL1, TP53, IKZF1, IDH1, FLT3, and TET1 (Figure B). Now this cell line, designated ZYXY-M2, is collected by China center for type culture collection (CCTCC).Discussion: Established cell lines are invaluable tools in cell biology. Right now, HEL and SET-2 are two cell lines that are most widely used in MPN-related researches. HEL is an erythroleukemia cell line which was derived from a patient with leukemia transformation of Hodgkin's disease, and SET-2 is a megakaryoblastic cell line which was derived from a patient with leukemic transformation of ET. Here, we established a cell line, ZYXY-M2, derived from a PMF patient and it would prove to be a valuable model for the study of PMF. ZYXY-M2 could help to explore the pathogenesis and development of PMF, novel drug screening, and may also help to set up xenograft PMF mouse models. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical and Laboratory Features of JAK2 V617F, CALR, and MPL Mutations in Malaysian Patients with Classical Myeloproliferative Neoplasm (MPN).

Mutations of JAK2V617F, CALR, and MPL genes confirm the diagnosis of myeloproliferative neoplasm (MPN). This study aims to determine the genetic profile of JAK2V617F, CALR exon 9 Type 1 (52 bp deletion) and Type 2 (5 bp insertion), and MPL W515 L/K genes among Malaysian patients and correlate these mutations with clinical and hematologic parameters in MPN. Mutations of JAK2V617F, CALR, and MPL were analyzed in 159 Malaysian patients using allele-specific polymerase chain reaction, including 76 polycythemia vera (PV), 41 essential thrombocythemia (ET), and 42 primary myelofibrosis (PMF) mutations, and the demographics of the patients were retrieved. The result showed that 73.6% JAK2V617F, 5.66% CALR, and 27.7% were triple-negative mutations. No MPL W515L/K mutation was detected. In ET and PMF, the predominance type was the CALR Type 1 mutation. In JAK2V617F mutant patients, serum LDH was significantly higher in PMF compared to PV and ET. PV has a higher risk of evolving to post PV myelofibrosis compared to ET. A thrombotic event at initial diagnosis of 40.9% was high compared to global incidence. Only one PMF patient had a CALR mutation that transformed to acute myeloid leukemia. JAK2V617F and CALR mutations play an important role in diagnostics. Hence, every patient suspected of having a myeloproliferative neoplasm should be screened for these mutations.

Results from Ongoing Phase 1/2 Trial of SL-401 in Patients with Myeloproliferative Neoplasms Including Chronic Myelomonocytic Leukemia and Primary Myelofibrosis

Background: SL-401 is a novel targeted therapy, comprised of recombinant IL-3 genetically fused to a truncated diphtheria toxin payload, directed to the interleukin-3 receptor α (CD123). CD123 is expressed by various cellular components of hematologic malignancies, including myeloproliferative neoplasms (MPN) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). CD123 is also expressed on immune plasmacytoid dendritic cells (pDCs), which have been shown to reside in the microenvironment of some malignancies, especially chronic myelomonocytic leukemia (CMML, 30-40%), where they may play a role in tumor growth. Accordingly, a therapy directed at CD123-expressing myeloid neoplastic cells and/or neighboring CD123-expressing pDCs could provide a therapeutic benefit. SL-401 is currently being evaluated in patients with MPN or MDS/MPN, including CMML and primary myelofibrosis (PMF). Preliminary results from this ongoing trial are reported here.Methods & Results: This is a multicenter, single-arm Phase 1/2 trial of patients with MPN receiving SL-401 who have relapsed or are unable to tolerate standard therapy. In Stage 1 dose escalation (enrollment completed), patients received SL-401 as a daily IV infusion at 7, 9, or 12 mcg/kg/day on days 1-3 of a 21, 28 or 42-day cycle (cycles 1-4, 5-7, >8, respectively) in a standard 3x3 design. In Stage 2 (ongoing), patients receive SL-401 at the dose determined in Stage 1. Primary objectives include characterization of the safety profile, including determination of the maximum tolerated or tested dose, and overall response rate (ORR). In Stage 1, no DLT or MTD was reached. The 12 mcg/kg dose was selected for Stage 2. Nineteen (19) patients received SL-401 (MF, n=11; CMML-1, n=5; CMML-2, n=2; systemic mastocytosis (SM), n=1), 13 of whom received SL-401 at the 12 µg/kg dose. The median age was 69 years (range: 42-81) with a median of 1 prior line of therapy. Median number of SL-401 cycles was 3 (range: 1-11+). The most common treatment-related adverse events (TRAEs) were fatigue (6/19; 32%), thrombocytopenia (5/19; 26%), anemia (5/19; 26%), hypoalbuminemia (4/19; 21%), and capillary leak syndrome (CLS) (4/19; 21%). CLS was reported in 4 patients: grade 2 (n=3) and grade 3 (n=1). All events resolved and patients remained on study. The most common ≥ grade 3 TRAEs were anemia (4/19; 21%) and thrombocytopenia (3/19; 15.7%). SL-401 monotherapy achieved a complete response (CR) in a CMML-1 patient (receiving SL-401 for 10+ months, ongoing). There were also 7 patients with stable disease (SD) (1-8 months duration), including in 1 PMF patient with pre-treatment platelet count <50,000. Sixty percent (60%; 3/5) of patients with SD who received SL-401 at the 12 mcg/kg dose experienced spleen reductions (of 46%, 29% and 21%) by physical exam. CD123 analyses and patient enrollment in Stage 2 are ongoing.Conclusions: SL-401 has been well-tolerated in patients with MPN and CMML, with primary TRAEs being fatigue, thrombocytopenia, anemia, hypoalbuminemia, and capillary leak syndrome (21% [4/19] including 1 grade 3 event). Early signs of activity include one CR (receiving SL-401 for 10+ months, ongoing), and 7 SDs (which include 3 spleen reductions of 21%-46% at the 12 mcg/kg dose). Given CD123 expression on myeloid neoplastic cells as well as pDCs within the microenvironment, SL-401 may offer a novel, targeted therapeutic approach in patients with certain MPNs of unmet medical need. CD123 analyses are ongoing and patient enrollment continues in Stage 2 of this Phase 1/2 trial. Clinical trial information: NCT02268253. DisclosuresGupta:Incyte: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Schiller:Celator/Jazz: Research Funding. Lee:Baxalta: Consultancy; Alexion Pharmaceuticals: Consultancy; Clinipace: Consultancy; Amgen: Consultancy. Yacoub:Novartis: Speakers Bureau; Sandoz: Consultancy; CTI: Consultancy; Pfizer: Consultancy; Seattle Genetics: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau. Talpaz:Pfizer Inc: Consultancy, Other: Travel, Research Funding; ARIAD: Other: Travel, Research Funding. Sardone:Stemline Therapeutics: Employment. Wysowskyj:Stemline Therapeutics: Employment. Shemesh:Stemline Therapeutics: Employment. Chen:Stemline Therapeutics: Employment. Brooks:Stemline Therapeutics: Employment, Equity Ownership, Patents & Royalties. Goswami:Stemline Therapeutics: Employment. Verstovsek:CTI BioPharma Corp: Research Funding; Bristol Myers Squibb: Research Funding; Galena BioPharma: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Astrazeneca: Research Funding; Genentech: Research Funding; Blueprint Medicines Corp: Research Funding; CTI BioPharma Corp: Research Funding; Incyte: Research Funding; Roche: Research Funding; Lilly Oncology: Research Funding; Seattle Genetics: Research Funding; Gilead: Research Funding; Astrazeneca: Research Funding; NS Pharma: Research Funding; Bristol Myers Squibb: Research Funding; Pfizer: Research Funding; Blueprint Medicines Corp: Research Funding; Incyte: Research Funding; Pfizer: Research Funding; Seattle Genetics: Research Funding; NS Pharma: Research Funding; Gilead: Research Funding; Promedior: Research Funding; Celgene: Research Funding; Promedior: Research Funding; Galena BioPharma: Research Funding; Roche: Research Funding; Lilly Oncology: Research Funding. Khoury:Kiromics: Research Funding; Pfizer: Research Funding; Stemline Therapeutics: Research Funding; Angle: Research Funding. Pemmaraju:abbvie: Research Funding; affymetrix: Research Funding; Incyte Corporation: Consultancy, Honoraria; cellectis: Research Funding; novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria; stemline: Consultancy, Honoraria, Research Funding; roche diagnostics: Consultancy, Honoraria.

JAK2, CALR, MPL, and ASXL1 Mutations in 136 Thai Patients with Philadelphia-Negative Myeloproliferative Neoplasms and Their Correlations with Clinical Outcomes

Background: Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPN) are a group of hematological malignancies, including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). Mutations of JAK2, CALR, MPL, and ASXL1 are associated with carcinogenesis and clinical characteristics of Ph-negative MPN. However, the availability of the data regarding these mutations is relatively limited in Thai population. Objective: To investigate these mutations in Thai Ph-negative MPN patients. Materials and Methods: One hundred thirty-six MPN (48 PV, 72 ET, and 16 PMF) cases were enrolled. Mutations of JAK2 V617F and MPL W515L/K mutations were investigated using allele-specific PCR (AS-PCR) and confirmed by sequencing. CALR and ASXL1 mutations were investigated using Sanger sequencing. Results: The JAK2 V617F mutation was detected in 83.3% of PV, 66.6% of ET, and 50.0% of PMF, and correlated with higher RBC, WBC, and PLT counts in PV. CALR mutations were detected in 16.7% of ET and 12.5% of PMF and associated with a higher PLT count in ET. The MPL W515L mutation was detected in one PMF patient. ASXL1 mutations were detected in 6.3% of PV, 8.3% of ET, and 12.4% of PMF, with c.1954G&gt;A being the preponderant mutational form. ASXL1 mutations increased the risk (RR 27.6) and accelerated the onset of AML transformation. Conclusion: The present study provided the prevalence and clinical correlation of JAK2, CALR, MPL, and ASXL1 mutations among Thai Ph-negative MPN patients. The association of ASXL1 mutations with adverse clinical outcomes suggested the potential usefulness of these mutations as a prognostic marker for Ph-negative MPN patients. Keywords: JAK2, MPL, CALR, ASXL1, Thai, Philadelphia-negative myeloproliferative neoplasm (MPN)

Mutated clones driving leukemic transformation are already detectable at the single-cell level in CD34-positive cells in the chronic phase of primary myelofibrosis

Disease progression of myeloproliferative neoplasms is the result of increased genomic complexity. Since the ability to predict disease evolution is crucial for clinical decisions, we studied single-cell genomics and transcriptomics of CD34-positive cells from a primary myelofibrosis (PMF) patient who progressed to acute myeloid leukemia (AML) while receiving Ruxolitinib. Single-cell genomics allowed the reconstruction of clonal hierarchy and demonstrated that TET2 was the first mutated gene while FLT3 was the last one. Disease evolution was accompanied by increased clonal heterogeneity and mutational rate, but clones carrying TP53 and FLT3 mutations were already present in the chronic phase. Single-cell transcriptomics unraveled repression of interferon signaling suggesting an immunosuppressive effect exerted by Ruxolitinib. Moreover, AML transformation was associated with a differentiative block and immune escape. These results suggest that single-cell analysis can unmask tumor heterogeneity and provide meaningful insights about PMF progression that might guide personalized therapy.

LSD1 Inhibition with IMG7289 (bomedemstat) Rebalances Megakaryopoiesis and Erythropoiesis in a Patient with MPN/MDS with Refractory Anemia with Ringed Sideroblast and Marked Thrombocytosis with JAK2 and SF3B1 Mutations

Lysine-specific demethylase-1 (LSD1) is a histone H3 K4 demethylase that plays a critical role in hematopoietic progenitor cell differentiation, in particular, the production of megakaryocytes and erythrocytes from their common classical bi-potent progenitor (MEP). LSD1 is recruited to chromatin by the transcription factor GFI1B to license the maturation of megakaryocyte-erythrocyte progenitor cells (MEPs). Anemia is a common and serious complication of primary myelofibrosis (PMF), which is often refractory to conventional therapy. We report here the effect of IMG-7289 (bomedemstat), an LSD1 inhibitor, on skewing the fate of MEPs favoring the production of red cells over platelets and producing a complete hematologic remission in a JAK2V617F-positive PMF patient who had developed a refractory anemia with ringed sideroblasts. A 63 year old woman presented in 2016 with a 1 year history of with fatigue significantly compromising her lifestyle. She denied fever, night sweats, or weight loss. The physical examination was unremarkable with no palpable splenomegaly. Laboratory studies showed a hemoglobin (Hb) of 11.3 g/dL, a total white blood cell count (WBC) of 20.3k/µL with leukoerythroblastosis with 1% circulating blast cells, and a platelet count of 1,192k/µL. A bone marrow biopsy (BMB) showed a hypercellular marrow with increased dysmorphic megakaryocytes but without dyserythropoiesis. There were no ringed sideroblasts with Perls stain, and silver staining showed grade 2 reticulin fibrosis. The karyotype was del(7)(q11.2) and genotyping identified a JAK2V716F allele (VAF not known). The patient was considered to have PMF. Initial treatment consisted of hydroxyurea but without any improvement in her symptoms. Financial constraints initially prevented access to ruxolitinib but the patient was eventually started on 15 mg BID in January 2018. She tolerated therapy well but discontinued therapy a year later owing to no improvement in her fatigue and opted for observation alone. Over the next year, her hemoglobin fell to the range of 9.0-9.5g/dl and she developed drenching night sweats. A BMB in August 2019 showed a hypercellular marrow with increased dysplastic megakaryocytes but now with 76% ringed sideroblasts. Reticulin stain showed grade 2-3 fibrosis. No other dysplastic features were observed. Additional genotyping was declined by insurance and the patient was considered to have an MPN/MDS overlap syndrome. Accordingly, the patient was enrolled in clinical trial IMG-7289-CTP-102 (NCT03136185), a study of the LSD1 IMG-7289. On entry, her Hb was 9.5 gm/dL with an absolute reticulocyte count of 129K/µL. The WBC was 22.7k/µL with 69% neutrophils and 11% monocytes. The platelet count was 1,585k/µL with a mean platelet volume (MPV) of 8.1 fL. Genotyping showed a JAK2V617F VAF of 27%, a new SF3B1K700E mutation with a VAF of 26%, and a DNMT3A mutation with a VAF of 95%. Spleen volume by imaging was 283.3 cm³. The starting IMG-7289 dose was 40 mg po QD. At week 12, the Hb rose to 13.8 g/dL while the absolute reticulocyte count fell to 40K/µL. The platelet count was 271k/µL with the MPV increasing to 10.5 fL. The WBC was 5.2k/µL with 58% neutrophils and 18% monocytes. The mutant JAK2 allele burden had dropped to 14%. The spleen volume was 223 cm³. The IMG-7289 dose was reduced to 35 mg QD after complaints of fatigue. At Week 24, Hb was 11.7 g/dL, WBC was 12.1k/µL and platelets were 743k/µL. The total symptom score (MPN SAF TSS) was reduced by 31% from baseline. The expected pharmacodynamic effects of LSD1 inhibition on hematopoiesis were evident in this patient: monocytosis with a decrease in neutrophils and a marked reduction in the platelet count. Most striking was the concurrent improvement in the patient's anemia which was associated with a persistent reticulocytosis present at the start of treatment but with a significant increase in red blood cells in the setting of a SF3B1 mutation. MEP fate decisions hinge on the balance between KLF1 and FLI1 with the former favoring an erythroid fate, the later, megakaryocytes. The transcription factor complex of FLI1, GATA1, FOG1 which recruits the LSD1-containing NuRD complex is essential for megakaryocyte function; IMG-7289 likely disrupts that complex favoring erythropoiesis over megakaryopoiesis. The inhibition of LSD1 may, therefore, in the setting of thrombocytosis and anemia, rebalance the fate of MEPs to ameliorate both abnormalities. Disclosures Yacoub: Novartis: Speakers Bureau; Agios: Honoraria, Speakers Bureau; Incyte: Speakers Bureau; Hylapharm: Current equity holder in private company; Cara Therapeutics: Current equity holder in publicly-traded company; Ardelyx: Current equity holder in publicly-traded company; Dynavax: Current equity holder in publicly-traded company; Roche: Other: Support of parent study and funding of editorial support. Rienhoff:Imago BioSciences: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties.

Clinical Analysis of 269 Ph Chromosome-Negative Myeloproliferative Neoplasms Patients Stratified by Age

To investigate the difference of clinical characteristics between young patients(age≤40 years old) and middle-older patients(age>40 years old) with the myeloproliferative neoplasms（MPN）. The clinical data (gene mutations， peripheral blood routine examinations， imaging examination and past history) of 269 MPN patients was collected and analyzed. In essential thrombocythemia (ET) group, the proportion of triple-negative type in young patients was higher than that in middle-older group, while the peripheral white blood cell（WBC） and platelets（PLT） counts in the first visit were lower. In polycythemia vera (PV) group, the total detection rate of JAK2V617F (80.65%) was lower than that of other research reports. Young patients with PV showed the lower JAK2V617F rate and lower WBC count, compared with the middle-older aged patients. Both CALR and MPL mutations were not found in PV patients. There was only 1 primary myelofibrosis (PMF) patient aged <40 years old. 91.67% of the patients merged splenomegaly and this rate was higher than that of ET or PV patients. It was found that there were a diagnosed familial MPN family and an undiagnosed family， and the youngest patient was only 8 years old. The second-generation gene sequencing detection for them was not carried out. Age is an important reference index in the assessment of risks. The MPN patients with different age and types show much difference in gene mutations, peripheral blood cell counts, thrombotic events and sizes of spleen. The onset ages of patients with familial MPN trends to be generational younger.

Molecular Characterization of Ph-Negative Myeloproliferative Neoplasms in Tumor Circulating Nucleic Acids

Introduction. Genetic studies in patients with Ph-negative myeloproliferative neoplasms (MPNs) are essential to establish a correct diagnosis and to optimize their management. Recently, it has been demonstrated that it is possible to detect molecular alterations present in solid tumors and hematologic neoplasms by the analysis of circulating tumor DNA in plasma samples, which is known as liquid biopsy. It has been reported that most of the circulating cell-free DNA (cfDNA) has its origin in immature hematopoietic and bone marrow cells; however, there is limited information about liquid biopsy applications in MPNs. Objective. To analyze the molecular profile of circulating tumor DNA in patients with MPNs. Patients and methods. Peripheral blood samples from 75 patients with MPNs were collected at the time of diagnosis: 21 polycythemia vera (PV), 42 essential thrombocythemia (ET), 10 primary myelofibrosis (PMF) and two non-classifiable MPNs. Cellular DNA was extracted from the granulocytic fraction isolated by density gradient centrifugation and cfDNA was obtained from 1-3ml of plasma (MagMAX Cell-Free DNA Isolation Kit, Thermo Fisher Scientific). cfDNA purity was ascertained by capillary electrophoresis (4200 TapeStation system, Agilent). Molecular characterization was performed in paired samples of granulocytes DNA and cfDNA by next generation sequencing (NGS). Libraries were prepared using a custom panel that covered the whole codifying region of 25 myeloid-associated genes (QIAseq Custom DNA Panels, Qiagen) and sequenced using Illumina technology (Miseq, Nextseq) with a 3000x minimum coverage. Results. The amount of total cfDNA/mL in plasma was significantly higher in PMF (mean 97 ng/ml) than in PV and ET (mean 18 and 23g/ml, respectively) (p = 0.003, Kruskal-Wallis). Overall, 144 mutations in driver (JAK2, CALR, MPL) and non-driver genes were detected in the granulocytic fraction with similar frequencies to what has been described for PV, ET and PMF. The most frequently mutated non-driver genes where ASXL1 (18.7%), TET2 (17.3%), DNMT3A (6.7%), SRSF2 (6.7%) and IDH2 (5.3%). Sequencing of cfDNA showed a total of 146 mutations. All mutations detected in the granulocytic fraction were also detected in the paired cfDNA sample (100% concordance); two additional mutations in MPL and ASXL1 were detected in plasma in one case. The median variant allele frequency (VAF) present in cfDNA was 29% (range 0.86 - 91.73%), which is far superior to what has been described in solid neoplasms or lymphomas (median 0.41%, range 0.03% - 97.6%). A strong correlation was observed between the VAFs of granulocytic DNA and cfDNA (r = 0.875, p &lt; 0.001, Spearman) (Figure 1). The mutation VAFs detected in cfDNA were significantly higher than VAFs detected in granulocytes (p &lt; 0.001, Wilcoxon). In particular, MPL mutations presented 2.5 higher VAF in cfDNA than in granulocytes (p = 0.018, Wilcoxon). This finding was confirmed and quantified by digital PCR. Interestingly, in one PMF patient the p.W515L MPL driver mutation was originally only detectable by NGS in cfDNA, but not in granulocytes. This mutation was identified by ultra-sensitive digital PCR in both cfDNA (VAF 2.30%) and granulocytes (VAF 0.16%). Conclusions. The analysis of circulating tumor DNA allows the characterization of the molecular abnormalities of patients with Ph negative myeloproliferative neoplasms. The sensitivity for mutation detection in driver and non-driver genes was equal or even superior to that obtained when studying the isolated granulocytic population. Disclosures Salar: Roche: Research Funding, Speakers Bureau; Janssen Pharmaceuticals: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy. Besses:Gilead: Research Funding. Bellosillo:TermoFisher Scientific: Consultancy, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau.

18F-FLT PET/MRI for bone marrow failure syndrome-initial experience

BackgroundBone marrow failure syndrome (BMFS) is a heterogeneous group of disorders associated with single- or multiple-lineage cytopenia and failure of normal hematopoiesis. We assessed the feasibility of integrated PET/MRI with 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) to assess the pathophysiology of whole-body bone marrow for the diagnosis and monitoring of BMFS. Twenty-five consecutive patients with BMFS underwent a pre-treatment 18F-FLT PET/MRI scan. They included 7 patients with aplastic anemia (AA), 16 with myelodysplastic syndrome (MDS), and 2 with myeloproliferative neoplasms (MPNs), primary myelofibrosis (MF), and secondary [post-essential thrombocythemia (post-ET)] MF. Two of the seven AA patients underwent a post-treatment scan. Eight of the 16 MDS patients who exhibited decreased 18F-FLT uptake in the pelvis were considered to have hypoplastic MDS (hypo-MDS). 18F-FLT PET and diffusion-weighted imaging (DWI) were visually and quantitatively evaluated.ResultsThe 18F-FLT uptake in the ilium was strongly correlated with bone marrow cellularity based on biopsy samples (ρ = 0.85). AA patients exhibited heterogeneously decreased uptake of 18F-FLT according to disease severity. Multiple 18F-FLT foci were observed in the proximal extremities, and they were in the central skeleton in severe AA patients. Post-treatment 18F-FLT PET scans of severe AA patients reflected the response of hematopoietic activity to treatment. MDS patients had marked 18F-FLT uptake in the central skeleton and proximal extremities, whereas hypo-MDS patients had heterogeneously decreased uptake, similar to that of non-severe AA patients. 18F-FLT PET and DWI were unable to predict the progression to leukemia for both MDS and hypo-MDS patients. A primary MF patient had slightly decreased 18F-FLT uptake in the central skeleton, but marked expansion of bone marrow activity to the distal extremities and high uptake of tracer in the extremely enlarged spleen (extramedullary hematopoiesis). In contrast, a secondary (post-ET) MF patient demonstrated marked bone marrow uptake, reflecting the hypercellular marrow with fibrosis. DWI revealed diffusely high signal intensities in both the primary and secondary MF patients.Conclusion18F-FLT PET can be used to noninvasively assess whole-body bone marrow proliferative activity and DWI may reflect the different aspects of bone marrow pathophysiology from 18F-FLT PET. 18F-FLT PET/MRI is useful for the diagnosis and monitoring of BMFS, except for the differentiation between non-severe AA and hypo-MDS, and the prediction of progression to leukemia.

Accumulation of JAK Activation-Loop Phosphorylation Promotes Type I JAK Inhibitor Withdrawal Syndrome in Myelofibrosis

Patients with myelofibrosis who are treated with the Type I ATP competitive JAK inhibitor ruxolitinib derive symptomatic benefit from improvement in cytokine-mediated symptoms, and show cytoreductive responses in disease manifestations, such as splenomegaly or leukocytosis. However, ruxolitinib treatment does not consistently reduce JAK2 mutant allele burden nor eradicate the disease. Secondly, when ruxolitinib is withdrawn there is rapid recrudescence of cytokine-mediated symptoms within days of treatment stopping and in rare cases this has led to a life-threatening “ruxolitinib discontinuation syndrome” characterized by acute relapse of disease symptoms, splenomegaly, worsening cytopenia, and a cytokine storm akin to septic shock. Paradoxically, Type I inhibitors can induce the accumulation of JAK activation loop phosphorylation for unknown reasons, despite blockade of kinase function and inhibition of STAT phosphorylation. Here, we developed a time-dependent assay in primary myelofibrosis samples to mimic ruxolitinib withdrawal syndrome and investigate a possible mechanism of action involving JAK2 activation loop phosphorylation.Methods: Primary peripheral blood de novo myelofibrosis samples were obtained prior to treatment with informed consent according to institutional guidelines (Stanford University Institutional Review Board No. 6453 or Board No. SACRB-APP-020). TF1.8 cells were cultured in RPMI with 10% fetal calf serum supplemented with 2 ng/ml of GM-CSF. SET-2 cells were cultured in RPMI with 10% serum. Phosphatase assays were performed with recombinant JAK2 kinase domain was mixed with recombinant PTP1B and JAK inhibitor. All antibodies were sourced from Cell Signalling Technologies. Unless otherwise stated, P values comparing two means were calculated using the two-tailed unpaired Student's t-test in Prism version 6 (GraphPad Software, Inc. La Jolla, CA).Results: We developed a time-dependent assay to mimic ruxolitinib withdrawal in primary JAK2V617F and CALR mutant myelofibrosis patient samples and observed striking activation of spontaneous STAT signaling in JAK2V617F samples after drug washout. Accumulation of ruxolitinib-induced loop phosphorylation was dose-dependent and correlated with rebound signaling and the presence of a JAK2V617F mutation. Ruxolitinib prevented dephosphorylation of a cryptic site involving Tyr1007/1008 in JAK2 blocking ubiquitination and degradation. We note that one patient with CALR mutant did not exhibit the same degree of STAT spontaneous withdrawal signalling compared to JAK2 V617F. Similarly, CALR mutant myelofibrosis cells showed an undetectable level of activated JAK2 Tyr1007/1008 phosphorylation in the presence of ruxolitinib. This is consistent with a number of emerging reports using CALR mutated models of myelofibrosis but needs to be confirmed in further patient samples.CHZ868 is a potent JAK2-biased Type II inhibitor that has efficacy in pre-clinical models of MPN at high nanomolar concentrations (250-500 nM) but its effects on withdrawal signalling have not been studied. In contrast to Type I inhibitors, CHZ868 did not prevent JAK2 activation loop dephosphorylation as shown by in vitro phosphatase assay. CHZ868 was tested in SET2 cells and primary JAK2 V617F patient samples and did not induce accumulation of JAK2 phosphorylation. Most importantly CHZ868 did not exhibit rebound JAK2 signaling upon washout and was superior in the eradication of JAK2 mutant positive progenitors purified from 14 myelofibrosis patient samples. Immunophenotypically, CD34+ progenitor cells from myelofibrosis patients most resembled CD34+CD38+CD45RA+CD123hi granulocyte-myeloid progenitors and lymphoid-restricted multipotential progenitors.Consistent with our signaling, chi-square test of 10 published cases of severe ruxolitinib-withdrawal syndrome noted a high frequency of JAK2 mutant cases (100%) compared to expected mutation frequencies (2 x 2 contingency table, P= 0.0203). In majority of cases, symptoms began within 72 hours.Conclusions: Type I inhibitor-induced loop phosphorylation acts as a pathogenic signaling node released upon drug withdrawal, especially in JAK2V617F patients. Ruxolitinib-withdrawal syndrome may be more frequent in JAK2 V617F mutant cases. [Display omitted] DisclosuresHughes:Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ross:Celgene: Research Funding; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria.

Clinical analysis on granulocyte colony stimulating factor mobilized allogenetic peripheral blood mononuclear cell transfusion for one patient with primary myelofibrosis in fibrosis stage

Objective To investigate the safety and efficacy of granulocyte colony stimulating factor mobilized allogenetic peripheral blood mononuclear cell (G-PBMNC) in treatment of patient with primary myelofibrosis (PMF) in fibrosis stage with high risk and unsuitable for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods In March 2016, a case of male PMF patient (age: 70 years old)in fibrosis stage with high risk admitted to Department of Hematology, Affiliated Hospital of Panzhihua University was selected as a research subject. Laboratory and imaging examinations were performed to determine the stage and risk of the disease, combined with his medical history and outpatient examination results. After admission, the patient was treated with danazol, thalidomide and prednisone orally, deep intramuscular injection of deferoxamine, subcutaneous injection of erythropoietin (EPO), and suspended red blood cell infusion. In April 2016, the patient received G-PBMNC from his daughter. General condition and treatment safety of patient were observed, and the blood routine test results, transfusion volume of suspended red blood cell and spleen size were monitored. Relevant examination results, diagnosis, treatment and curative effect of this patient were analyzed retrospectively, and related literature was reviewed. This study protocol was approved by the Ethics Committee of Affiliated Hospital of Panzhihua College (No. 11 of the Ethics Examination in 2015), and informed consent was obtained from participant. Results ① After admitted, results of blood routine examination showed that white blood cell count was 2.0×109/L, red blood cell count was 1.5×1012/L, hemoglobin (Hb) value was 39.0 g/L, platelet count was 23.0×109/L, ratio of primitive cells in peripheral blood was 2%. Serum EPO value was more than 774 mIU/mL, and ferritin value was more than 2 250 μg/L. No specimens were taken from the bone marrow puncture. Result of bone X ray radiography showed bone sclerosis. Result of upper abdomen color Doppler ultrasound showed splenomegaly and slight hepatomegaly. The patient was diagnosed as PMF in fibrosis stage with high risk. ② A total of 140 mL of G-PBMNC suspension was isolated from the donor, and transfused to the patient after separation and counting. Number of mononuclear cell (MNC) and CD34+ cell were 3.0×108/kg and 1.6×106/kg, respectively, and acute graft versus host disease (aGVHD) did not develop 1 months after G-PBMNC infusion. ③ Most of the blood routine examination indexes of the patient showed an upward trend after G-PBMNC infusion. 1 and 11 months after G-PBMNC infusion, median white blood cell counts of the patient were 4.0×109/L (3.1×109/L-4.6×109/L) and 3.6×109/L (2.7×109/L-4.0×109/L), and were higher than that of 1.8×109/L (1.3×109/L-2.3×109/L) before G-PBMNC infusion. 8 and 13 months after infusion, median values of Hb were 69.7 g/L (59.2-82.5 g/L) and 70.5 g/L (61.3-84.1 g/L), and were higher than that of 46.8 g/L (33.5-60.3 g/L) before infusion. 6, 11 and 12 months after infusion, median platelet count were 36.5×109/L (22.6×109/L-43.6×109/L), 41.0 ×109/L (29.9 ×109/L-56.7 ×109/L) and 39.0×109/L (25.7×109/L-50.3×109/L), and were also higher than that of 14.0×109/L (7.2×109/L-18.5×109/L) before infusion . ④ Within one month before G-PBMNC infusion, a total of 12 U of suspended red blood cells were transfused; while one month after G-PBMNC infusion, amount of suspended red blood cell infusion decreased to 6.5 U. 3-4 months after infusion, transfusion volume of suspended red blood cell decreased to 1.5 U. 11 months after G-PBMNC infusion, transfusion volume of suspended red blood cell was only 3.5 U, and the patient did not receive blood transfusion for one month. ⑤ There was no progressive enlargement of spleen after infusion of G-PBMNC. Conclusions G-PBMNC could safely improve the routine blood test indexes of PMF patients in fibrosis stage with high risk and reduce the dependence of blood transfusion, so G-PBMNC could be a safe and effective treatment option for fibrotic PMF patients in no condition to allo-HSCT. But this conclusion is limited to the clinical analysis of a single case, and it requires large sample, multi-center, randomized controlled trials for further study and verification. Key words: Allogeneic cells; Transplantation, homologous; Hematopoietic stem cells; Primary myelofibrosis; Therapeutics

Successful engraftment after cord blood transplantation from an HLA‐homozygous donor (homo‐to‐hetero cord blood transplantation) in a primary myelofibrosis patient with broad HLA antibodies

Donor-specific human leukocyte antigen (HLA) antibodies are a significant risk factor for graft failure in cord blood transplantation (CBT). Although there are several treatments to decrease HLA antibodies, such as platelet transfusion, plasma exchange, rituximab, and bortezomib, their effectiveness has not been established. We herein report the case of a primary myelofibrosis (PMF) patient with broad HLA antibodies who underwent CBT from an HLA-homozygous donor in which the alleles were matched only in the host-versus-graft direction (homo-to-hetero CBT). The cord blood was killer cell immunoglobulin-like receptor (KIR) ligand matched. She received a reduced-intensity conditioning regimen. We used tacrolimus and mycophenolate mofetil as prophylaxis against graft-versus-host disease (GVHD). The neutrophils engrafted on Day 31. A chimerism analysis with fluorescence in situ hybridization of peripheral blood cells showed 99.9% donor type on Day 33. She developed only mild acute skin GVHD and chronic skin GVHD. This case indicates the usefulness of homo-to-hetero CBT in a patient with broad HLA antibodies with a strong mean fluorescence intensity, which is a significant risk factor for graft failure. Further studies are necessary to determine the risk of GVHD and to elucidate the association between KIR ligand incompatibility and graft failure in homo-to-hetero CBT.

Characterization of a human induced Pluripotent Stem (iPS) cell line (INCABRi002-A) derived from a primary myelofibrosis patient harboring the 5-bp insertion in CALR and the p.W146X mutation in TP53

Primary myelofibrosis (PMF) is a hematological malignancy characterized by activation of the JAK/STAT pathway and risk of leukemic transformation. In this study, we generated an induced Pluripotent Stem (iPS) cell line derived from a 65-year old male PMF patient carrying the 5-pb insertion in the CALR gene (CALRins5) and the c.437 G > A mutation in the TP53 gene (p.W146X). The newly derived PMF3.17 iPS cell line harbors the original mutations and was characterized as bona fide iPS.Resource tableUnlabelled TableUnique stem cell line identifierINCABRi002-AAlternative name(s) of stem cell linePMF3.17InstitutionBrazilian National Cancer Institute and D'Or Institute for Research and EducationContact information of distributorMartin Bonamino, PhD, mbonamino@inca.gov.br, Bárbara Monte Mór, PhD, barbara.montemor@inca.gov.br, Stevens Rehen, PhD, srehen@lance-ufrj.orgType of cell lineiPS cellOriginHumanAdditional origin infoAge: 65Sex: maleCell sourcePeripheral bloodClonalityClonalMethod of reprogrammingSendai VirusGenetic modificationNoType of modificationNot applicableAssociated diseasePrimary myelofibrosisGeneCALR gene: CALRins5, c.1154_1155insTTGTC, p.K385 fs*47TP53 gene: c.437 G > A, p.W146XMethod of modificationNot applicableName of transgene or resistanceNot applicableInducible/constitutive systemNot applicableDate archived/stock dateJuly 7th, 2015Cell line repository/bankNot applicableEthical approvalEthics Committee of the Brazilian National Cancer Institute (INCA) under the number 062/08. Ethics Review Board-competent authority obtained.

Abstract 3093: Genomic characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident

Abstract Philadelphia-chromosome negative chronic myeloproliferative neoplasms (MPNs) are a unique group of hematological malignancies including Polycytemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) characterized by impaired function and structure of bone marrow. MPN driver mutations are usually found in Janus Kinase 2 (JAK2), Thrombopoietin Receptor (MPL) and Calreticulin (CALR) genes, however, 10-15% of MPN cases are triple negative (TN) for these mutations. A previous study showed a lower rate of JAK2 V617F mutations in PMF patients exposed to sublethal doses of ionizing radiation (IR) from the Chernobyl accident in Ukraine. We hypothesized a lower rate of the usual driver mutations in IR-exposed MPN patients. To examine whether there are distinct driver mutations, 281 Ukrainian IR-exposed and unexposed MPN patients, were enrolled in the study. Their records were reviewed for classification by the WHO 2016 MPN criteria. Genomic DNA was obtained from the peripheral blood leukocytes of 281 MPN patients to identify JAK2 V617F, MPL W515, and type 1- and 2-like CALR mutations by allele-specific PCR, Sanger Sequencing and RT-PCR, respectively. Copy number alterations and copy-neutral loss of heterozygosity (cnLOH) were assessed in 30 PMF patients by high-density Affymetrix CytoScan HD oligo-SNP microarray platform. Whole exome sequencing was used to identify additional genetic variants in these 30 PMF patients. Statistical significance for categorical variables and continuous variables were evaluated by Fisher's exact test and Wilcoxon's rank sum test using Statistical Analysis R, version 3.4.2. Clinical features of exposed and unexposed MPN patients were similar. More PMF IR-exposed patients were transfusion dependent (32.4%) than PMF unexposed patients (14.1%) (p = 0.04). JAK2 V617F was detected in 58% of IR-exposed and in 74% of unexposed MPN patients (p = 0.007). JAK2 V617F was also less frequent in IR-exposed PV patients, but not statistically significant. Type 1-like CALR mutation was detected in 12% of exposed and 3% of unexposed patients (p = 0.033). Overall, exposed patients were TN in 28% of IR-patients versus 16% of unexposed patients (p = 0.027). Among other genetic variants, ATM S1691R mutation with cnLOH at 11q22.3 was identified in one TN IR-exposed PMF patient. Previously the mutation was reported, but not in MPN patients. Missense mutations in EZH2 at 7q36.1 and SUZ12 at 17q11.2 with copy number loss were also identified in TN IR-exposed PMF patients. Our results confirm a lower rate of JAK2 V617F and higher rate of TN cases among IR-exposed MPN patients versus unexposed. We also demonstrated a higher frequency of type 1-like CALR mutation in IR-exposed MPN patients and new potential MPN driver mutations. Thus, IR-exposed MPN patients represent a disease group with distinct genomic characteristics worthy of further study. Citation Format: Larysa Poluben, Maneka Puligandla, Donna Neuberg, Christine R. Bryke, Nancy Hsu, Sergiy Klymenko, Olga Mishcheniuk, Oleksandr Shumeiko, Steven Balk, Xin Yuan, Olga Voznesensky, German Pihan, Miriam Adam, Ernest Fraenkel, Paula G. Fraenkel. Genomic characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3093.

Abstract 954: TGF-ß signaling inhibition counteracts myelofibrosis in MPN

Abstract Myelofibrosis (MF) is a common feature of the Philadelphia-Chromosome negative (Ph-) myeloproliferative neoplasms (MPN), a group that includes Primary Myelofibrosis (PMF), Essential Thrombocytosis (ET) and Polycythemia Vera (PV). Current treatment with Jak2 inhibitors reduces splenomegaly, but does not eliminate malignant clones nor improve bone marrow (BM) or spleen fibrosis, underlining the need for alternative therapies. One promising target is TGF-β1, a cytokine crucial for the development of myelofibrosis that is elevated in PMF patient plasma, progenitors and megakaryocytes (MK), and is known to promote fibrosis through stimulation of fibroblasts and their progenitors, mesenchymal stroma cells (MSC). Dysregulation of TGF-β1 signaling has been described in a number of mouse models of myelofibrosis exhibiting aberrant megakaryopoiesis, one of the hallmarks of MPNs. Preliminary data from 93 MPN patients showed overexpression of TGF-ß in PMF samples (p=0.001), with increased activation of the canonical target SMAD2 (p=0.005). We tested the efficacy of the orally available TGF-β1 signaling inhibitor Galunisertib (LY2157299) in counteracting fibrosis in two MF mouse models of common MPN oncogenes Jak2 and Mpn. Mice in the first model express the constitutively active human Jak2V617F mutant under the control of the ubiquitous hematopoietic promoter vav1 and show a PV-like phenotype at 6 weeks old, progressing to fibrosis of bone marrow (BM) and spleen around 25-30 weeks. Thirty week old mice were treated with Galunisertib for 4 weeks and showed a significant decrease in fibrosis compared to control mice. Similar results were observed in 50 week old mice treated with a murine antibody against TGFβRII (IMC-TR1,LY3022859) for 4 weeks.Our second model relies on transplantation of mice with BM cells transduced with MPLW515L, a mutant mpl variant that is active in the absence of TPO. These mice rapidly develop MF, myeloproliferation and splenomegaly. Flow cytometry showed an expansion of immature and mature megakaryocytes with overexpression of TGF-β1 in MPLW515L cells. Treatment from day 12 to day 26 after transplantation reduced both white blood cell counts (a measure of myeloproliferation) and fibrosis as measured by reticulin staining. In vitro, Galunisertib counteracted the pro-fibrotic effects of TGF-ß1 on both murine and human MSC by antagonizing its stimulatory effects on collagen I and III production. Analysis of MSCs from Galunisertib-treated mice also showed reduced collagen I and III production compared to controls. In conclusion, we offer further evidence that aberrant megakaryopoiesis drives TGF-β1 overproduction in MPN by increasing megakaryocyte numbers and overexpressing TGF-β1 in mutant MKs. Galunisertib counteracts TGF-ß1 induced fibrosis in two MPN mouse-models and is a promising candidate for symptomatic treatment of myelofibrosis. Citation Format: Matthias Bartenstein, Lanzhu Yue, Wanke Zhao, Wanting Tina Ho, Cem Murdun, Adam W. Mailloux, Ling Zhang, Anjali Budhakoti, Kith Pradhan, Franck Rapaport, Huaquan Wang, Zonghong Shao, Ulrich Steidl, Ross L. Levine, Zhizhuang Joe Zhao, Amit K. Verma, Pearlie K. Epling-Burnette. TGF-ß signaling inhibition counteracts myelofibrosis in MPN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 954. doi:10.1158/1538-7445.AM2017-954

Efficacy of ALK5 inhibition in myelofibrosis

Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-β family members are profibrotic cytokines and we observed significant TGF-β1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-β1 was also observed in murine clonal MPLW515L megakaryocytic cells. TGF-β1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-β receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPLW515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPLW515L and JAK2V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-β/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF.

CXCL12/CXCR4 pathway is activated by oncogenic JAK2 in a PI3K-dependent manner

JAK2 activation is the driver mechanism in BCR-ABL-negative myeloproliferative neoplasms (MPN). These diseases are characterized by an abnormal retention of hematopoietic stem cells within the bone marrow microenvironment and their increased trafficking to extramedullary sites. The CXCL12/CXCR4 axis plays a central role in hematopoietic stem cell/ progenitor trafficking and retention in hematopoietic sites. The present study explores the crosstalk between JAK2 and CXCL12/CXCR4 signaling pathways in MPN. We show that JAK2, activated by either MPL-W515L expression or cytokine stimulation, cooperates with CXCL12/CXCR4 signaling to increase the chemotactic response of human cell lines and primary CD34+ cells through an increased phosphatidylinositol-3-kinase (PI3K) signaling. Accordingly, primary myelofibrosis (MF) patient cells demonstrate an increased CXCL12-induced chemotaxis when compared to controls. JAK2 inhibition by knock down or chemical inhibitors decreases this effect in MPL-W515L expressing cell lines and reduces the CXCL12/CXCR4 signaling in some patient primary cells. Taken together, these data indicate that CXCL12/CXCR4 pathway is overactivated in MF patients by oncogenic JAK2 that maintains high PI3K signaling over the threshold required for CXCR4 activation. These results suggest that inhibition of this crosstalk may contribute to the therapeutic effects of JAK2 inhibitors.