Primary Myelofibrosis Pts Research Articles

Plitidepsin Inhibits the Growth of Cells Harboring JAK2V617F Mutation.

Abstract 3907Poster Board III-843Plitidepsin (Aplidin®) is a novel cyclic depsipeptide derived from the marine tunicate Aplidium albicans, currently obtained by chemical synthesis, that is under Phase II clinical development. Plitidepsin is effective against a large panel of tumor cells, and although precise action mechanisms have still to be ascertained the drug induces an oxidative stress, activation of Rac1 GTPase and inhibition of protein phosphatases, overall leading to sustained activation of JNK and p38MAPK. In a previous report (Verrucci M et al, ASH 2008, 2787A) we evaluated Plitidepsin activity in the GATA-1low murine model of myelofibrosis. Plitidepsin corrected thrombocytopenia of myelofibrotic mice, reduced the frequency of megakaryocytes (Mk) and normalized angiogenesis in the bone marrow, and prevented extramedullary hematopoiesis. In the present study, we assessed the effects of Plitidepsin on cell lines harboring homozygous (HEL and UKE-1, a gift of W. Fiedler) or heterozygous (SET2) JAK2V617F mutation and on cells from patients (pts) with myeloproliferative neoplasms (MPN). In a short-term (3 days) proliferation assay we found that Plitidepsin prevented cell growth with IC50 values of 1.0±0.3 nM for HEL, 0.5±0.03 nM for UKE-1, and 0.8±0.02 nM for SET2, that were all lower than 1.5±0.1 nM for the BCR/ABL mutated K562 cell line (P<.001 in case of UKE-1 cells). Also Ba/F3 cells transduced with the V617F allele (a gift of R. Skoda) were found more sensitive to Plitidepsin (IC50= 0.03±0.01 nM) than the wild-type counterpart (IC50= 0.4±0.03 nM; P<0.02). Similar results were obtained using a 14-day clonogenic assay in agar cultures. These data indicated that Plitidepsin was active at very low nanomolar concentrations against cell lines harboring JAK2V617F mutation. We then evaluated the effects of Plitidepsin on the growth of BFU-E, CFU-GM and CFU-Mk from MPN pts; all five Polycythemia Vera (PV) and 4/5 Primary Myelofibrosis (PMF) pts analyzed were JAK2V617F mutated. As shown in the Table, PMF pts presented significantly lower IC50 value than controls (Ctrl; P<.002) for all type of clonogenic progenitors; cells from PMF pts resulted also significantly more sensitive to Plitidepsin than those from PV patients (P<.02), while the difference between PV and Ctrl did not reach the significance level. To evaluate whether Plitidepsin also affected the latest stages of differentiation and maturation of MKs, that is the most overtly affected cell lineage in PMF, we added Plitidepsin on day +7 of a two-stage liquid culture system initiated with CD34+ cells purified from the PB of PMF patients; the generation of CD61+ Mks was measured 5 days later by FACS analysis. However, we found that the number of CD61+ cells was no different between cultures containing or not Plitidepsin, overall suggesting that the drug mainly affected early proliferation of Mk progenitors rather than influencing their differentiation. We then performed single colony genotyping to quantify the proportion of hematopoietic colonies harboring the JAK2V617F mutation which grew in growth factor-supplemented methylcellulose cultures initiated with purified CD34+ cells from PMF patients in the presence of 1 nM Plitidepsin. Initial data in 3 pts were available; in one, the proportion of JAK2-mutated BFU-E colonies decreased from 51% to 27% while no changes were observed in the other two pts. Finally, since a correlation between levels of p27(Kip1) and the response of tumor cells to Plitidepsin has been described, we measured p27 levels in different cell lines after exposure to Plitidepsin. We observed that p27 mRNA levels increased 15-fold and 30-fold in UKE1 and HEL cells, respectively, compared to K562 cells after 24 hr with 1nM Plitidepsin; such an increase was mirrored by a protein content 1.9- to 3.5-fold greater than baseline in UKE-1 cells at 1 and 10 nM Plitidepsin, suggesting that JAK2V617F mutated cells responded to the drug by modulating their p27 levels. Collectively, we provided evidence that Plitidepsin has in-vitro activity against MPN cells, particularly from PMF pts. These results, as well as those which were previously described in the GATA1low murine model, provided the rationale for a clinical trial in patients with myelofibrosis that is being developed within the Myeloproliferative Disorders Research Consortium (MPD-MRC).Plitidepsin IC50 (nM)BFU-ECFU-GMCFU-MkCtrl (n=5)8.7 ± 2.38.2 ± 3.51.7 ± 0.9PV (n=5)5.2 ± 2.07.4 ± 4.0not donePMF (n=5)1.1 ± 0.61.6 ± 0.40.4 ± 0.06 Disclosures:Aracil:PharmaMar: Employment. Fe Paz:PharmaMar: Employment. Vannucchi:PharmaMar: Research Funding.

The Relationship Between Chromosomally Abnormal Hematopoiesis and the JAK2V617F Allele Burden in Patients (pts) with Ph-Negative Chronic Myeloproliferative Disorders (Ph-neg MPD)

The JAK2V617F mutation, present in >90% of pts with polycythemia vera (PV) and in approximately 50% of pts with primary myelofibrosis (PMF) and essential thrombocythemia (ET), has been identified in CD34+ cells from pts with PV and PMF, consistent with the notion that this mutation arises in an early hematopoietic progenitor cell (HPC). The relationship between the recurrent chromosomal abnormalities, occurring occurring in 8% of ET, 29% of PV and ~50% of PMF pts, to the JAK2V617F mutation is uncertain. In order to determine a relationship between the JAK2V617F allele burden and the frequency of HPC with chromosomal abnormalities, we identified 5 pts (3 PV, 1 PMF and 1 ET) who were JAK2V617F positive and had different cytogenetic abnormalities. At the time of the study Pt 1 (PMF) had +9, del(20)(q11q13) in 50% of cells and del(20)(q11q13),+der(9)t(1;9)(q21;q13) in 50% cells. Pt 2 (PV) had 85% of cells with +9. Pt 3 (PV) had 15% of cells with del(20)(q11q13). Pt 4 (ET) had 36% of cells with dup(1)(q21q31) and Pt 5 (PV) had +der(9)t(1;9)(q21;q13) in 55% cells. The JAK2V617F allele burden in granulocytes and CD34+ cells ranged from 55% to >95%. HPC (BFU-E, CFU-GM and CFU-Meg) were assayed from CD34+ cells and each individual colony was plucked, split in half, and tested for both the JAK2V617F mutation and the presence of the marker cytogenetic abnormality using FISH. Among the 289 colonies studied, 82% were JAKV617F homozygous, 8% were heterozygous for JAK2V617F and 10% contained only wild type (WT) JAK2. Cytogenetically abnormal colonies, containing the marker chromosome, as detected by FISH, were observed in 80% of the JAK2V617F homozygous, 64% of JAK2V617F heterozygous and 30% of the WT JAK2 colonies. Despite 100% of the colonies being homozygous for JAK2V617F in Pt 3 only 7% of colonies exhibited del(20q). In Pt 1, with two distinct cytogenetically abnormal populations, 2% of JAK2V617F homozygous colonies contained del(20q) alone, while 40% of colonies had both del(20q) and +9p and 52% had a combination of all three abnormalities. Further analysis of Pts 2 and 5 demonstrated that 87% and 47%, respectively, of JAK2 homozygous colonies contained gain of 9/9p. Duplication (1)(q21q31), a characteristic chromosomal abnormality of Pt 4, was identified in 18% of JAK2 WT colonies while its frequency in homozygous JAK2V617F HPC was 76%. These observations suggests that an increased JAK2V617F allele burden is associated with an increased frequency of cytogenetically abnormal HPC. A small proportion of homozygous JAK2 V617F HPC are involved by del(20q) (2%–7%) indicating their low proliferative capacity. Acquisition of trisomy 9 or trisomy 9p either in the presence of del(20q) or alone, involved a much larger population of JAK2V617F homozygous HPC (61%–93%) suggesting their higher proliferative capacity. In colonies containing only WT JAK2, 12% to 26% of cells within colonies had either +9 or dup(1q) suggesting that these chromosomal abnormalities might occur independently of JAK2V617F. These studies suggest a complex relationship between the JAK2V617F allele burden and occurrence of clonal chromosomal abnormalities in Ph-neg MPD HPC which might define important biological differences and clinical outcomes in MPD.

Improved Survival in Red Blood Cell Transfusion Dependent Patients with Primary Myelofibrosis (PMF) Receiving Iron Chelation Therapy.

Patients (pts) with PMF and iron overload (IOL) may receive iron chelation therapy (ICT), although there are no data demonstrating that this improves clinical outcome. Red blood cell (RBC) transfusion dependent (TD) pts with thalassemia receiving ICT have improved survival and decreased end-organ toxicities and RBC-TD pts with myelodysplastic syndrome (MDS) receiving ICT have improved survival. We performed a review of 41 pts seen from January 1987 to April 2007 with a bone marrow biopsy confirmed diagnosis (Dx) of PMF. Clinical data were collected from the practice database, the Provincial Home Hemosiderosis Program of British Columbia database, and by chart review. Pts receiving ICT were treated with desferrioxamine (DFO) 0.5–3g by subcutaneous infusion over 12 hours, 35 days per week or with deferasirox (DFX) 20mg/kg/day orally, dose adjusted to response and pt tolerance. 29 were male and 12 female. Median age at PMF Dx was 64 (43–86) years (y) and 24 pts were >60y. White blood cell (WBC) count at Dx was <4 or >30x109/L in 8, hemoglobin (Hgb) <100 G/L in 5, platelet count <100x109/L in 5, monocyte count >1x109/L in 7. Karyotype analysis was: normal, n=16; del(6)(q25), n=1; tri(14), str12p, n=1; complex, n=1. Lille, Strasser and Mayo prognostic scores were: low risk, n=15, 8, 11; intermediate, n=15, 19, 9; high, n=5, 11, 5 respectively. Primary PMF treatment was: supportive care, n=23; hydroxyurea, n=10; immunomodulatory, n=4; splenectomy, n=2. Clinical evidence of IOL was documented in 21 pts; number of RBC units (NRBCU) received, n=18; ferritin >2000ug/l, n=6 (and ferritin >1000ug/l, n=1); CHF, n=5; liver disease, n=3; endocrine, n=3. 16 pts were RBC transfusion–independent (TI) and 25 were TD; of these 10 received ICT. Median duration of ICT was 18.3 (0.1–117) months (mo) and reasons for initiating ICT were: NRBCU received, n=9; elevated ferritin, n=6; clinical evidence of IOL, n=3. Five pts received DFO, 4 DFX, and 1 DFO followed by DFX. In ICT pts, initial/Pre-ICT ferritin levels were significantly higher than in TD-NO ICT pts at a median of 2318 (range 263–8400) and 527 (120–934) mg/L respectively (p=0.05) and decreased significantly in TD-ICT pts at most recent follow-up to 1571 (1005–3211) mg/L (p=0.01). Causes of death were: TI patients, no deaths; TD-NO ICT patients, 11 deaths (73%): probably PMF-related, n=9; progression to PMF-blast phase (BP), n=3; sepsis, n=3; cardiac, n=2; bleeding, n=1, unknown, n=2 ; TD-ICT patients, 2 deaths (20%); PMF-BP, n=1; bleeding, n=1. Kaplan- Meier analysis showed a median overall survival (OS) for all pts of 126.5 (14.4–293.2) mo. In a univariate analysis of TD pts, factors significant for OS (and 5y OS) were: WBC count at Dx (4.0–30x109/L, 69%; <4.0 or >30x109/L, 0%; p=0.002); monocyte count at Dx (<1.0x109/L, 74%; >1.0x109/L, 0%; p=0.0001); Mayo prognostic score (low, 67%; intermediate, 50%; high, 0%; p=0.05); NRBCU transfused (<20U, 30%; 21–50U, 27%; >50U, 12%; p=0.02) and receiving ICT (ICT, 89%; NO-ICT, 34%; p=0.003). In Cox regression analysis of TD pts, factors significant for OS were: NRBCU (p=0.001) and ICT (p=0.0001). For TI, TD-NO ICT and TD-ICT pts respectively the median OS was not reached (NR) at 200 mo, 58 mo and NR at 293 mo respectively (p=0.01 for TD-NO ICT vs TI and NS for TD-ICT vs. TI). The hazard ratio (HR) for pts receiving >20 RBCU was increased at 7.6 (95% CI 1.2–49.3) and the HR for pts receiving ICT was improved at 0.15 (0.03–0.77). In conclusion, 61% of PMF pts developed RBC-TD and had inferior OS, however TD pts receiving ICT had superior OS compared to TD pts not receiving ICT and the OS of TD-ICT pts was comparable to the OS of TI patients, suggesting a benefit to ICT. These are to our knowledge the first data documenting improved clinical outcome in pts with PMF receiving ICT. Prospective studies of IOL and the impact of ICT in pts with PMF are warranted.

Hypermethylation of CXCR4 Promoter, and Its Reactivation by Hypomethylating Agent, in CD34+ Cells from Primary Myelofibrosis Patients

Abnormal mobilization of CD34+ hematopoietic stem/progenitor cells in peripheral blood (PB) characterizes patients (pts) with Primary Myelofibrosis (PMF), and is attributed to both disruption of CXCR4/SDF-1 axis by a bone marrow (BM) proteolytic environment (Xu M, Blood 2005) and reduced CXCR4 expression on CD34+ cells (Rosti V, BCMD 2007) due to a transcriptional defect (Guglielmelli P, Stem Cells 2007). CXCR4 expression on CD34+ cells was inversely related to their number in PB and associated with advanced disease. We aimed at addressing potential mechanisms of reduced CXCR4 transcription in PMF CD34+ cells. To rule out cytokine-induced transcriptional regulation, CD34+ cells from controls and PMF pts were exposed to a range of SDF-1, G-CSF, TGF-beta and Interferon-gamma concentrations, and CXCR4 protein and mRNA level were concurrently measured by flow cytometry and quantitative PCR (RTQ-PCR), respectively. SDF-1 at 500 ng/mL for 24 hrs reduced CXCR4+/CD34+ cells to less than 10% basal value while CXCR4 mRNA level was unchanged, suggesting ligand-induced receptor internalization but no effect on gene transcription. Other cytokines tested were ineffective. Since abnormal methylation of CXCR4 has been implicated in reduced CXCR4 expression in pancreatic cancer cells (Sato N, Canc Biol Ther 2005), we evaluated methylation pattern of five CpG islands in CXCR4 promoter in PMF CD34+ cells and in HEL, K562 and HL-60 cell lines as control, using both methylation-specific PCR and bisulphite sequencing. A correlation was found between CXCR4 expression and extent of CpG island methylation in cell lines: K562 had 8±6% CXCR4+ cells and all five CpG islands were methylated while in HL-60, that showed bright CXCR4 expression in 90% of the cells, the promoter was in a completely un-methylated state; in HEL cell line, that expressed CXCR4 in 37±10% of cells, partial methylation of CpG island 1 and 5 was observed. On the other hand, CXCR4 promoter CpG island 1 hypermethylation was found in CD34+ cells of 15 PMF pts, and in a minority of them also of CpG island 4 or 5. The effect of hypomethylating agent 5-azacytydine (5-AZA; 1 uM up to 72 hrs) was evaluated in CD34+ cells from 10 PMF pts; starting from 12 hr incubation, significantly increased proportion (from 3- to greater than 10-fold basal level) of CD34+ cells expressed CXCR4, and a three- to five-fold induction of CXCR4 RNA by RTQ-PCR was detectable at 8–12hr. Similar effects were observed after 5-AZA treatment of HEL, while in K562 cells changes in CXCR4 expression were minimal. Concurrent incubation with 5-AZA and HDAC inhibitor SAHA induced effects similar to hypomethylating agent alone. In both PMF CD34+ and HEL cells treated with 5-AZA, hypermethylation of CpG 1 was almost completely reverted at 24–48 hr. Finally, 5-AZA treated CD34+ cells showed enhanced migration in response to SDF-1. These data point to abnormal methylation of CXCR4 promoter as a mechanims for reduced CXCR4 expression in PMF CD34+ cells, possibly contributing to their constitutive migration in PB. Additionally, owing demonstration of an effect of 5-AZA combined with HDAC inhibitor TSA on the proportion of JAK2617V>F PMF mutant clones in culture (Shi J, Canc Res 2007), they represent a proof-of-principle for exploring use of hypomethylating agents in PMF.

Transfusion Dependence in Patients with Primary Myelofibrosis Has a Negative Impact on Survival Independent of Decreased Myelopoiesis.

Primary myelofibrosis (PMF) is a myeloproliferative disorder characterized by progressive bone marrow failure, extramedullary hematopoiesis and risk of progression to blast phase (BP). Many PMF patients (pts) require red blood cell (RBC) transfusions, risking iron overload (IOL)-related organ dysfunction. Pts with myelodysplastic syndrome and RBC transfusion dependence (TD) have worse overall survival (OS), which may be improved by iron chelation therapy (ICT). To assess the effect of TD and ICT on survival in PMF, we reviewed 30 pts seen from 1985 to 2007 with a marrow biopsy confirmed diagnosis. 21 pts were male and 9 female and median age at Dx was 66.5 (range 43–84) years (y). ECOG Performance Status; 0, n=13; 1, n=9; 2, n=7; 3, n=1. WBC count was <4.0 or >30x109/L at diagnosis in 5 pts, and hemoglobin (Hb) <100G/L in 14. Karyotype in 13 pts: normal, n=11; del (6)(q25), n=1; complex, n=1. Lille Prognostic Score was: 0, n=10; 1, n=11; 2, n=4. 20 pts were RBC-TD with total units (U) transfused: 1–20, n=6; 21–50, n=3; >50, n=11. Initial PMF treatment was: supportive care, n=21; low-dose chemotherapy, n=4; immunomodulatory, n=3; splenectomy, n=2. Clinical evidence of IOL, n=14 total: CHF, n=4; liver disease, n=3; endocrine, n=3; ferritin ≥1000 ug/L at Dx, n=6. Baseline features that differed between transfusion-independent (TI) and TD and between non-ICT-TD and ICT pts: total RBCU transfused (p=0.0001 and p=0.03) and evidence of IOL (p=0.003 and p=0.06), respectively. 5 pts received ICT for a median of 75.7 (range 2.9–117) months (mo); 4 received desferrioxamine (DFO) 0.5–3g by subcutaneous infusion 12 h/d, 5 d/wk and 2 received deferasirox (1 switched from DFO). At a median follow-up (FU) of 58.8 (0.1–243.7) mo, 2 non-ICT pts and 1 ICT pt progressed to PMF-BP; 2 received chemotherapy and all 3 died of progressive BP within weeks. Median OS for all pts, TI, TD and ICT pts was: 102.1 (14.4–243.7) mo; not reached at 204.9 mo; 60.8 (14.4–243.7) mo and 83.6 (60.8–202.9) mo and 5y OS was 67%; 100%; 55% and 66% respectively (p=0.014 for TD vs. TI). Factors significant for OS were: RBC-TD (p=0.014, hazard ratio [HR] 43.6, confidence intervals [CI] 41–46.2); increasing RBC transfusion requirement (TR; 2 fold change in RBCU/4wk; p=0.018, HR 5.0 [4.3–5.7]); Hb<100G/L (p=0.03, HR 3.8, [3.2–4.4]); total RBCU transfused (p=0.0001, HR 1.3 [1.0–1.6]). 12 TD pts (60%) died: cardiac, n=3; infection, n=3; bleeding, n=2 (1 ICT pt); PMF-BP, n=3; unknown, n=1. Initial/pre-ICT mean ± standard error (se) ferritin was 2337±1038ug/L in ICT pts and 506±466ug/L in non-ICT pts, and FU ferritin decreased in ICT pts to 1902±428ug/L (p=0.03) and was 824±445ug/L in non-ICT pts (p=NS). Initial and FU neutrophil counts (NC) in ICT pts were a mean ±se of 4.7±1.6 and 5.0±1.7 vs. 7.2±1.5 and 20.6±7.7 x109/L in non-ICT pts; platelet counts (PLTC) were 432±89; 527±208; 293±50; and 203±36 x109/L respectively and RBCU transfused/4 wk were 2.3±0.6; 3.3±0.8; 1.3±0.3; 1.8±0.4 (p=NS for all and for TI vs. TD NC and PLTC). In conclusion, 67% of pts with PMF developed RBC-transfusion dependence, which portended worse OS. However, there was no significant decrease in neutrophil or platelet counts or increase in RBC transfusion requirements. This suggests the effect of TD on OS was not from impaired myelopoiesis alone, and that there may be an impact of IOL secondary to TD on outcome. Prospective studies of ICT in PMF pts are warranted.