ALBERT

Description: Abstract 794FN2 LBH589 is a novel pan-deacetylase inhibitor (DACi) that has demonstrated clinical activity in phase I/II studies in patients with a variety of hematologic malignancies. Our group has previously presented preliminary results of a phase I study of LBH589 in patients with myelofibrosis (MF) (Mascarenhas et al, ASH 2009, a308) while a phase II trial using higher doses of LBH589 has also been reported (DeAngelo et al, ASH 2010,a630). Both studies identified reversible thrombocytopenia as the DLT and reported evidence of clinical responses. The final results of our phase I study and the effects of extended treatment with LBH589 are reported here. We enrolled 18 patients at 3 dose levels. Fifty-five percent of these patients had PMF, 28% Post-PV MF and 17% Post ET MF; all were intermediate/high risk based on Lille classification. Twenty-five mg PO TIW was determined to be the recommended phase II dose. All patients experienced resolution of their systemic symptoms and 10/11 patients with baseline palpable splenomegaly, who were evaluable after 1 month of therapy, had a median reduction of 30%, range 0–100%. Five patients entered into an extension phase of the trial and received 〉 6 months of therapy with a mean dose of 20mg PO TIW at time of optimal response (Table 1). Of these patients, 2 were initially enrolled in the 20 mg PO TIW cohort, 1 in the 30 mg PO TIW cohort and 2 in the 25 mg PO TIW cohort. Both patients at the lowest dose achieved clinical improvement (CI) by IWG-MRT response criteria at 6 months as did one patient at the 25 mg dose. The remaining 2 patients had SD at 30 and 25 mg. A mean reduction in palpable splenomegaly at 3 and 6 months of 55% and 83%, respectively, was observed in this group. Two of these patients had marked and durable improvement in anemia (patients 1 and 4). Patient 4 achieved a near CR at 16 months with resolution of palpable splenomegaly, elimination of peripheral blood dacrocytes and leukoerythroblastosis, a 4g/dL increase in hemoglobin, improvement in overall marrow cellularity and megakaryocyte atypia with an increase in erythroid precursors and a significant reduction of reticulin/collagen fibrosis. Patient 1 was heavily transfusion dependent requiring RBC transfusions weekly to maintain a mean hemoglobin of 6.5g/dL and after 6 months on LBH589 achieved 〉50% reduction in transfusion dependence maintaining a mean hemoglobin of 9g/dL. Patient 2 had resolution of palpable splenomegaly and leukoerythroblastosis by cycle 6 and the bone marrow at cycle 26 was characterized by a reduction in marrow fibrosis from grade 4 to 1. A phase II study is ongoing, 14 patients are currently enrolled, with a planned goal of 22 patients. Pharmacokinetic and pharmacodynamic studies as well as cytokine profiling of the phase I patients are being analyzed and will be presented at the meeting. We conclude that low doses of LBH589 delivered for greater than 6 months in patients with MF are capable of ameliorating symptoms, improving clinical features and reversing pathologic marrow changes. Disclosures: Off Label Use: Oral histone deacetylase inhibitor that targets epigenetic changes in malignant myelofibrosis cells with an goal to modify the disease process.

Description: Treatment of polycythemia vera (PV) with the Murine Double Minute 2 (MDM2) antagonist, idasanutlin, in a phase 1 trial was reported by our group to be well tolerated with a high overall response rate (Mascarenhas et al, Blood. 2019 Jun 5). A global, phase 2 trial is currently underway evaluating idasanutlin in hydroxyurea (HU) resistant/intolerant PV patients (NCT03287245). MDM2, a negative regulator of TP53 is upregulated in PV CD34+ cells and inhibition of MDM2 targets PV hematopoietic stem/progenitor cells (HSPC) (Lu et al, Blood. 2014;124(5):771-90). Additional trials of MDM2 antagonists have shown promise, however, there is concern that these agents have the potential to induce TP53 mutations or promote expansion of TP53 mutated clones. Resistance to MDM2 inhibitors has been evaluated in solid tumor cell lines and attributed to either the emergence of de novoTP53 mutations or the selection of TP53 mutated clones. (Michaelis et al, Cell Death Dis. 2011;2:e243; Skalniak et al, Cancers. 2018;10(11)). The effect of MDM2 inhibition on TP53 mutant clones is of particular interest in myeloproliferative neoplasms (MPNs). TP53 mutations have been reported with a low allele burden in ~15% of chronic MPN patients (Kubesova et al, Leukemia. 2018;32(2):450-61), however, TP53 loss of heterozygosity and rapid expansion of TP53 mutant clones is associated with transformation to blast phase (Lundberg et al, Blood 2014,123:2220-8). As reported, in the idasanutlin PV trial, 1/13 patients were identified to have a baseline pathogenic TP53 mutation in hematopoietic cells (VAF 5.5%), using a deep sequencing assay with a limit of detection (LOD) of VAF 0.5%. This patient was a non-responder to idasanutlin and upon treatment had an increasing JAK2V617F and TP53 mutant VAF. End of study hematopoietic cell specimens of all patients were deep sequenced (LOD 0.1%) and revealed that 4 additional patients harbored detectable TP53 mutations after idasanutlin treatment with VAF ranging from 1-12%. In each sample, 1-5 unique TP53 mutations were identified, all within the hotspot domain of the TP53 gene. Deeper sequencing of baseline and follow-up samples revealed these mutations were present at a subclonal level (VAF 0.1-5.5%) and increased over time, indicating that treatment with the MDM2 antagonist promoted expansion of already existing TP53 mutant clones (Table 1, Figure 1). None of the patients who lacked a TP53 mutation at baseline developed a TP53 mutant clone with idasanutlin treatment. There was no clear association of presence of TP53 mutations with prior HU, anagrelide or interferon exposure. There has been careful monitoring of patients to determine whether the expanding TP53 clone has clinical ramifications. Patients were on study for a median of 54 weeks (23-131). The only patient who exemplified resistance to idasanutlin was the single patient with a high burden TP53 mutation (37%). All other patients were taken off study due to patient choice/toxicity. Furthermore, all TP53 mutant and non-mutant patients have had stable disease with no evidence of progression to MF or AML. Sequencing of 2 patients post-discontinuation of idasanutlin revealed that the VAF of the TP53 mutant clones decreased since the agent was discontinued. Updated patient molecular data post-treatment discontinuation will be reported at the meeting. To investigate whether idasanutlin induces de novo TP53 mutations in PV myeloid cells we performed long term HSPC cultures. Mononuclear cells from 6 distinct PV patients were treated continuously with idasanutlin (500 nM) over ~6 weeks and DNA from both treated and untreated colonies were analyzed using next generation sequencing with a LOD of 2% VAF and no TP53 mutations were detected. The combined in vitro and clinical data reveals that treatment with an MDM2 antagonist is not associated with the emergence of de novoTP53 mutations but rather the expansion of prior existing TP53 clones. This does not appear to have clinical repercussions, however, close monitoring of these patients is essential. We recommend that patients be screened for TP53 mutations prior to treatment with an MDM2 antagonist and that if present the TP53 mutant VAF be followed during their treatment course. Resistance to MDM2 inhibition is likely dependent on the TP53 mutant VAF and further studies will need to clarify the ideal dosing schedule of MDM2 antagonists and/or combinatorial therapy to prohibit TP53 mutant clonal expansion. Disclosures Houldsworth: Cancer Genertics: Other: stock in; Sema4: Employment. Rossi:Sema4: Employment. Kiladjian:AOP Orphan: Honoraria, Research Funding; Celgene: Consultancy; Novartis: Honoraria, Research Funding. Rampal:Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy; Constellation, Incyte, and Stemline Therapeutics: Research Funding. Mascarenhas:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche: Consultancy, Research Funding; Merck: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Promedior: Research Funding; Merus: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees. Hoffman:Merus: Research Funding.

Description: Abstract 2865 We have recently reported that MDM2 levels are increased in PV CD34+ cells as a consequence of JAK2617F, while the p53 levels are reduced in CD34+ cells from both patients with PV and PMF. These observations led us to explore therapeutic approaches by which to up-regulate p53 as a strategy to treat MPN patients. We previously reported that combination treatment with low doses of Peg IFNα 2a and Nutlin-3, an antagonist of MDM2, induced PV CD34+ apoptosis and inhibited PV colony formation significantly. The combination of these agents also decreased the proportion of JAK2V617F-positive HPCs. We demonstrated that these two drugs affect p53 through different pathways with Peg IFNα 2a activating p38 MAP kinase and STAT1 leading to increased p53 transcription while nutlin-3 prevents the degradation of p53 (Lu et al, Blood, 2012; In Press). RG7112 is an orally bioactivity small molecule inhibitor of p53-MDM2 binding which activates the p53 pathway. RG7112 is currently being evaluated in several phase 1 cancer trials. Based on our previous data, we hypothesized that RG7112 would be an effective drug to combine with low doses of IFN alpha to treat MPN patients. In order to evaluate the therapeutic effects of RG7112 alone or in combination with Peg IFNα 2a on hematopoietic progenitor cells from MPN patients, CD34+ cells were isolated from patients with PV and PMF patients and were treated with varying of doses of RG7112 alone or/and in combination with a low dose of Peg IFNα 2a and its effects on PV CD34+ cell proliferation, apoptosis and colony formation was evaluated. The effects of this drug on MPN CD34+ cells was compared to its effects on cord blood CD34+ cells After treatment with RG7112 at doses ranging from 100 nM to 5000 nM for 4 days, the numbers of PV CD34+ cells were reduced to a far greater degree (about 60%) than that observed with cord blood CD34+ cells (90% and 80%) at the doses between 200 and 500 nM indicating that low doses of RG7112 selectively affects MPN HPCs. 200nM of RG7112 and 200 U/ml of Peg-IFNα 2a alone induced cell cycle arrest of PV CD34+ cells, and combination treatment significantly promoted apoptosis of MPN CD34+ cells (2.5 folds). This observation leads us to examine the possibility that low doses of RG7112 might be used in combination with Peg-IFNα 2a to treat MPN patients. Treatment with 200 nM of RG7112 alone decreased PV CFU-GM and BFU-E-derived colony formation by 20% while combination treatment with 200 U/ml of Peg-IFNα 2a decreased PV CFU-GM and BFU-E-derived colony formation by 60% and 70%, respectively. Similarly, treatment with 200 nM of RG7112 alone decreased PMF CFU-GM and BFU-E-derived colony formation by 20% and 40%, respectively, while combination treatment with 200 U/ml of Peg-IFNα 2a decreased PMF CFU-GM and BFU-E-derived colony formation by 60% and 80%, respectively. We also found that treatment with both agents in combination also decreased the size of the hematopoietic colonies. By contrast, treatment with the same low doses of RG7112 and Peg-IFNα 2a alone or in combination did not significantly affect the ability of normal CD34+ cells to generate CFU-GM and BFU-E-derived colonies. Combined treatment with low doses of RG7112 and Peg-IFNα 2a decreased the total number of JAKV617F-positive PV HPC by 9% - 25% (p=0.014) leading to an increased proportion of colonies with wild type JAK2. In conclusion, these data demonstrate that IFNα plus RG7112 specifically target malignant MPN HPC by activating p53 in vitro, which we propose can be exploited therapeutically to eliminate JAK2V617F positive HPC in both PV and PMF patients. These results strongly suggest that combinations of low doses of IFNα and RG7112 might serve as a novel therapeutic strategy for the long term treatment of MPN patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1752 Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by abnormal trafficking of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC), resulting in their constitutive mobilization and the establishment of extra-medullary hematopoiesis. At present, there is no known therapeutic approach capable of altering the natural history of MF, except for allogeneic stem cell transplantation. Treatment with JAK2 inhibitors has been shown to lead to a rapid and dramatic reduction of splenomegaly although having only a modest effect on the JAK2V617F allele burden and not resulting in the elimination of cytogenetic abnormalities or correction of histopathological abnormalities (Verstovsek S, et al. N Engl J Med. 2010; 363:1117-27). To date, the mechanism underlying the reduction of splenomegaly observed following the treatment with a JAK2 inhibitor remains the subject of speculation. Recently, we observed the presence of MF-stem cells (MF-SC) in the spleens of MF patients and demonstrated that these splenic MF-SCs have a distinct differentiation program that distinguishes them from MF peripheral blood (PB) counterparts (Wang X, et al. Journal of Clinical Investigation, 2012. In Press). We therefore explored the effect of a JAK2 Inhibitor, AZD1480, on splenic MF-SCs in order to provide an explanation for the dramatic effects on MF spleen size. Treatment of splenic or PB MF CD34+ cells with cytokines+AZD1480 (CAZD, 150nM) for 3 days resulted in a significant reduction in the number of total cells, CD34+ cells and assayable HPC (CFU-GM, BFU-E and CFU-Mix) as compared with splenic or PB MF CD34+ cells treated with cytokines alone (CA), respectively (P all

Description: Myelofibrosis (MF) is characterized by anemia, thrombocytopenia and marrow fibrosis. Malignant rather than normal hematopoietic progenitor cells (HPC) preferentially proliferate. A form of MF can also occur in patients with a prior history of PV or ET which is referred to as PV or ET related MF (PV-MF and ET-MF). In MF, excessive proliferation of marrow stromal cells occurs in response to cytokines elaborated by the malignant clone. Previously, we found that lipocalin-2 (LCN2) levels were elevated in patient plasmas with MPN, and a greatest degree of elevation was observed in the PMF plasma and PMF conditioned medium. We also found that LCN2 increased PMF HPC proliferation and increased numbers of JAK2V617F homozygous HPCs, but depressed normal BM HPC (Lu. et al, 2013 ASH meeting). In this study, a series of experiments were performed to further explore the effects of LCN2 on MF pathogenesis. ELISA data showed that plasma LCN2 levels not only were higher in all MF patients, plasma LCN2 levels in PV-MF as well as ET- MF were significantly greater than that in PV or ET plasma (p=0.0002 and p=0.0175, respectively). Importantly, in MPN patients, a non-parametric spearman correlation coefficient analysis suggested an inverse relationship between the patients’ hemoglobin levels and LCN2 levels (coefficient=-0.35, p

Description: The TP53 pathway has been shown to be dysregulated in myeloproliferative neoplasms (MPN) through several different mechanisms, including TP53 mutations, TP53 deletions and Murine Double Minute 2 (MDM2) overexpression. Downregulation of the TP53 pathway likely plays a role in MPN progression as evidenced by the association of TP53 loss of heterozygosity with transformation to acute leukemia and the presence of inactivating mutations of TP53 found in a proportion of MPN-blast phase (MPN-BP) cases (Kubesova et al, Leukemia. 2018;32(2):450-61). Furthermore, MDM2 inhibition induces TP53 pathway upregulation and consequent targeting of hematopoietic stem and progenitor cells in polycythemia vera (PV) and myelofibrosis (MF). The MDM2 inhibitor, idasanutlin, has shown activity in a Phase I trial of high-risk PV patients (Mascarenhas et al. Blood. 2019 Jun 5). An additional regulator of TP53 is protein phosphatase, Mg2+/Mn2+ dependent 1D (PPM1D) which by dephosphorylating TP53 and stabilizing MDM2 regulates the DNA damage response pathway and apoptosis. Somatic activating mutations of PPM1D are present in both solid and hematologic malignancies and are specifically associated with therapy-related myeloid disorders (Hsu et al. Cell Stem Cell. 2018 Nov 1;23(5):700-13). Grinfeld et al. (N Engl J Med. 2018 Oct 11;379(15):1416-30) recently reported that in their genomic analysis of 2035 MPN patients, PPM1D was the eighth most common mutated gene in MPNs at 1.9% frequency. To determine whether there was an association of PPM1D mutations with MPNs we analyzed the genomic data of patients who participated in several clinical trials executed by the Myeloproliferative Neoplasm Research Consortium (MPN-RC). Of 89 MPN-BP patients, 5 patients harbored a PPM1D mutation with a median variant allele frequency (VAF) of 17%. In comparison, 4 out of 135 high risk PV and ET patients harbored a PPM1D mutation with a median VAF of 24%. All mutations were truncating which is consistent with previous reports of PPMID mutations in malignancies. The PPM1D gene is located on the long arm of chromosome 17 (17q23.2) and we hypothesized that cytogenetic aberrations involving this gene locus might also contribute to abnormalities of PPM1D function. Through analysis of our cytogenetic database, 16/1,124 (1.4%) MPN patients were found to have cytogenomic abnormalities involving the region containing the PPM1D gene. Eight of these patients had karyotypic abnormalities, including 3 pts with isochromosome 17q resulting in a gain of 17q and a loss of 17p, including the TP53 gene. The other 4 patients had structural variations of 17q which might lead to aberrant expression of PPM1D. One patient by FISH analysis had gain of 17q. Ten patients had cytogenomic aberrations of 17q detected through analysis of array comparative genomic hybridization and single nucleotide polymorphism (aCGH+SNP), 2 of which had concurrent karyotypic abnormalities of 17q. All patients had a gain or copy neutral loss of heterozygosity (cnLOH) of the 17q22-24.2 region. CnLOH of this region could lead to aberrant overexpression of the PPM1D gene. One of the 8 patients with cnLOH of 17q harbored a PPM1D mutation. Of the 16 patients with 17q cytogenomic abnormalities, 7 (44%) had MPN-BP and 7 (44%) patients had ET or ET that progressed to myelofibrosis (MF) or MPN-BP indicating an association of these abnormalities with advanced disease. By quantitative real time-PCR we determined the PPM1D transcript levels in mononuclear cells from 31 MPN patients without known PPM1D mutations (7 polycythemia vera (PV), 6 ET, 14 MF, 4 MPN-BP) and compared the transcript levels to mononuclear cells from healthy control patients. Forty-two percent (13/31) patients had overexpression of PPM1D (〉1.5 fold increase range). Fold increase ranged from 1.5-8 and overexpression was present in the following diagnoses: 4/7 PV, 1/6 ET, 8/14 MF and 0/4 MPN-BP. We have provided evidence that a number of abnormalities of PPM1D including activating mutations, cytogenetic aberrations and transcript overexpression are present in a significant proportion of MPN patients. These abnormalities in PPM1D can be additional events that lead to the downregulation of TP53 and contribute to MPN disease progression. We propose that inhibitors of PPM1D may be used in combination with other drugs that upregulate TP53 to treat MPN patients. Disclosures Rampal: Constellation, Incyte, and Stemline Therapeutics: Research Funding; Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy. Mascarenhas:CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche: Consultancy, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Promedior: Research Funding; Merus: Research Funding. Hoffman:Merus: Research Funding.

Description: Introduction: P53 is negatively regulated by MDM2 and increased expression of MDM2 in PV and MF CD34+ cells and megakaryocytes has been observed. Recently, we have shown that MDM2 antagonists termed nutlins can selectively eliminate MPN CD34+ cells (1-2). We have reported a phase 1 trial of PV patients treated with an oral nutlin, RG7388, which resulted in hematological responses and an improvement in symptoms in a substantial subset of PV patients intolerant or refracting to frontline therapy (3). Nutlins, however only target malignant cells with wild type p53, cannot completely eliminate the MPN stem cell pool and are associated with gastrointestinal toxicity. We therefore searched for another class of compounds to combine with nutlins to more effectively eliminate MF stem cells. Recently, an oral imipridone compound, ONC201, has been evaluated in clinical trials of patients with hematological malignancies (leukemia/lymphoma) and solid tumors. ONC201 was discovered by screening drug libraries for compounds that transcriptionally induced the TNF-related apoptosis inducing ligand (TRAIL) which triggers apoptosis through the extrinsic pathway which is independent of p53. ONC201 is also as potent allosteric agonist of mitochondrial caseinolytic protease P (ClpP) (4). Hyperactivation of ClpP by ONC201 increases mitochondrial proteolysis and leads to mitochondrial dysfunction and triggers the extrinsic apoptotic pathway. We hypothesized that combination treatment with nutlin (RG7112/RG7388) and ONC201 might provide an effective therapeutic strategy to selectively induce apoptosis of MF CD34+ cells by activating both intrinsic and extrinsic apoptosis pathways. Results: We tested whether RG7112 combined with ONC201 was capable of specifically targeting MF CD34+ cells. Treatment with ONC201 alone decreased total hematopoietic colonies generated by MF CD34+ cells in a dose dependent fashion. Treatment with ONC201 at dose of 10 uM decreased MF colony numbers by 50%, and a 500nM dose of RG7112 decreased MF colony numbers by 30%. Combination therapy with RG7112 and ONC201, however, decreased MF colony numbers by 70% (ONC201 vs RG-ONC201: p=0.005 and RG vs RG-ONC201: p=0.001, respectively). By contrast, treatment with these agents alone or in combination did not decrease colony formation by normal CD34+ cells. ONC201 or RG7112 alone at the doses utilized did not alter the genotype of the MF colonies; however, combination therapy decreased the numbers of JAK2V617F+ colonies by 20%, and increased wild type colony numbers by 24%. These data suggest that combination treatment with RG7112 and ONC201 can specifically deplete MF malignant CD34+ cells. We next tried to understand the mechanism by which combination therapy with RG7112 and ONC201 specifically targeted MF CD34+ cells. We found increased transcript levels of ClpP in untreated MF CD34+ cells as compared to normal CD34+ cells (p=0.03). Exposure of MF CD34+ cells to this drug combination significantly induced apoptosis of MF CD34+ cell (p=0.04), but did not induce apoptosis of normal CD34+ cells. Treatment of MF CD34+ cells with ONC201 alone increased transcript levels of TRAIL and death receptor 5 (DR5), but treatment with RG7112 alone did not have a similar effect. Furthermore, a combination of RG7112 and ONC201 synergistically increased not only TRAIL and DR5 but also ClpP transcript levels to a far greater extent. Treatment with RG7112 and ONC201, however, alone or in combination did not elevate ClpP, DR5 or TRAIL transcripts level in normal CD34+ cells. Treatment of MF CD34+ cells with either RG7112 or ONC201 alone increased TRAIL and DR5 protein levels to a modest degree but the degree of elevation was far greater with a combination of these two drugs. Treatment with RG7112 and ONC201 alone or in combination also increased FOXO3a, ClpP and ATF4 protein levels which are the upstream regulators of TRAIL and DR5, respectively (Figure 1). Summary: These data indicate that ONC201 combined with RG7112 is a potentially effective strategy for the treatment of MF. This drug combination selectively targets MF CD34+ cells by activating different apoptotic pathways that are both p53 dependent and p53 independent. Such a therapeutic approach would be anticipated to promote the elimination of both p53 wild type and p53 mutated MPN stem cell clones resulting in a greater depletion of MF but not normal stem cells. Disclosures Hoffman: Merus: Research Funding.

Description: Key Points Combination treatment with RG7112 and Peg-IFNα 2a targets primitive JAK2V617F+ progenitor cells in myeloproliferative neoplasms.

Description: Interferon (IFN-α) is effective therapy for polycythemia vera (PV) patients, but it is frequently interrupted because of adverse events. To permit the long-term use of IFN, we propose combining low doses of IFN with Nutlin-3, an antagonist of MDM2, which is also capable of promoting PV CD34+ cell apoptosis. Combination treatment with subtherapeutic doses of Peg IFN-α 2a and Nutlin-3 inhibited PV CD34+ cell proliferation by 50% while inhibiting normal CD34+ cells by 30%. Combination treatment with Nutlin-3 and Peg IFN-α 2a inhibited PV colony formation by 55%-90% while inhibiting normal colony formation by 22%-30%. The combination of these agents also decreased the proportion of JAK2V617F-positive hematopoietic progenitor cells in 6 PV patients studied. Treatment with low doses of Peg IFN-α 2a combined with Nutlin-3 increased phospho-p53 and p21 protein levels in PV CD34+ cells and increased the degree of apoptosis. These 2 reagents affect the tumor suppressor p53 through different pathways with Peg IFN-α 2a activating p38 MAP kinase and STAT1, leading to increased p53 transcription, whereas Nutlin-3 prevents the degradation of p53. These data suggest that treatment with low doses of both Nutlin-3 combined with Peg IFN-α 2a can target PV hematopoietic progenitor cells, eliminating the numbers of malignant hematopoietic progenitor cells.

Description: Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm (MPN) characterized by preferential proliferation of malignant hematopoietic progenitor cells which leads to excessive proliferation of marrow microenvironmental cells which are not involved by the malignant process. These events result in a clinical disorder characterized by anemia, a leukoerythroblastic blood picture, constitutive mobilization of CD34+ cells, extramedullary hematopoiesis, dacrocytosis, marrow megakaryocytic hyperplasia, progressive splenomegaly and reticulin and collagen marrow fibrosis. Several cytokines elaborated by PMF hematopoietic cells including TGF-b, vascular endothelial growth factor and tumor necrosis factor a have been implicated as playing a role in creation of the MF clinical phenotype. Neutrophil gelatinase associated Lipocalin-2 (LCN2) has been reported to have two distinct roles in the pathobiology of chronic myeloid leukemia (CML), suppressing residual normal HPC development and promoting CML proliferation (Devireddy LR et al, 2005, Cell). We, therefore hypothesized that LCN2 might also play a role in the development of the phenotypic features of PMF. Plasma LCN2 levels were measured by ELISA in 77 patients with PMF and were shown to be elevated as compared to 16 normal plasmas (P