Lü, Min

Introduction: Elevated pro-inflammatory cytokines are a hallmark feature of myeloproliferative neoplasms (MPNs). The pro-inflammatory cytokine interleukin-8 (IL8) is increased in patients with myelofibrosis (MF) and correlates with adverse outcome, including overall survival. Previously, the Levine/Fang labs identified increased IL8 secretion from individual CD34+ stem cells in a subset of MF patients. The role of IL8 and its cognate receptors CXCR1/2 in MF pathogenesis has not been delineated. Methods: Single-cell cytokine assays were performed on isolated CD34+ cells from 60 clinically annotated MPN patients (20 MF, 20 PV, 20 ET) using a previously described micro-chip platform (Kleppe et al, Can Disc 2013). 10 healthy donors (CD34+ cells from hip replacements) were used as controls. Integrated RNA-Seq and Assay for Transposase-Accessible Chromatin followed by next-generation sequencing (ATAC-Seq) was performed on CD34+ cells from MPN patients with and without expanded IL8 secreting clones for gene expression/chromatin accessibility analysis. To model the role of IL8-CXCR2 on fibrosis in vivo, the human MPLW515L transplant model (hMPLW515L) of MF was used. Specifically, wild-type (WT) murine bone marrow (Creneg-Cxcr2f/f; Cxcr2WT) or marrow lacking the CXCR2 receptor (VavCre-Cxcr2f/f; Cxcr2KO)were retrovirally infected with MSCV-hMPLW515L-IRES-GFP and transplanted into lethally irradiated WT recipient mice and monitored for disease. Blood counts, chimerism, and flow cytometry were assayed. Moribund mice were sacrificed and assayed for grade reticulin fibrosis and overall survival. Results: Single-cell cytokine assays confirmed an increased proportion of IL8-secreting CD34+ cells in MF patients (40%) in comparison to other MPN sub-types (10% PV/0% ET) (Figure 1A). MF patients with expanded IL8 secreting clones (defined as >50% of total CD34+ cells) had increased leukocytosis (p<0.0001), larger spleen sizes (p=0.0004), greater prevalence of constitutional symptoms (p=0.0084), and higher-grade reticulin fibrosis in marrow (Figure 1B) in comparison to MF patients without prevalent IL8 clones. IHC confirmed increased IL8 expression in marrow biopsies from 8/15 MF patients in comparison to 0/4 normal controls (Figure 1C), and high IL8 expression was also observed in MF splenic megakaryocytes (MKs) as well as in splenic stromal/endothelial cells not seen in normal spleen (Figure 1D). Integrated RNA-Seq/ATAC-Seq analysis of IL8-high MF patients confirmed up-regulation of IL8-CXCR2 signaling and enrichment in pro-inflammatory pathways (i.e TNFa, NFkB, etc) by GSEA, as well as increased expression/accessibility of pro-inflammatory genes S100A8 and S100A9-previously implicated in fibrosis development. Flow analysis of IL8-high MF CD34+ cells revealed enhanced surface expression of CXCR2 and its analog CXCR1, such that MF was characterized by increased IL8 ligand and receptor expression (Figure 1E) and coincided with enhanced NFkB pathway activity (Figure 1F). Consistent with this, colony forming assays of cultured MF CD34+ cells revealed enhanced colony output when cultured with IL8 compared to WT CD34+ cells-an effect ameliorated by co-treatment with the CXCR1/2 antagonist Reparixin (Figure 1G). In vivo, hMPLW515L adoptive transplant with Cxcr2KO hematopoietic donor cells demonstrated improved leukocytosis, thrombocytosis (Figure 2A) and splenomegaly in comparison to Cxcr2WT hMPLW515L recipient mice. Pathologic analysis revealed a reduction in reticulin fibrosis in bone marrow (Figure 2B) and spleen, translating into an improvement in overall survival (Figure 2C). Notably, a significant reduction in dysplastic MKs-a hallmark feature of MF-was also observed in Cxcr2KO hMPLW515L mice (Figure 2D) supporting a role for CXCR2 signaling in MK proliferation. Conclusion: IL8 secreting clones are associated with increased symptom severity and fibrosis grade in MF. Gene expression of MF CD34+ IL8 secreting clones shows up-regulation of inflammatory genes S100A8/A9, implicated in myofibroblast proliferation. Cxcr2 KO abrogates fibrosis formation and prolongs survival in the hMPLW515L model, and CXCR1/2 inhibition impairs colony forming capacity of MF CD34+ cells. These data suggest pharmacologic inhibition of this pathway should be investigated as potential therapy in MF and in PV/ET patients at high risk of fibrotic transformation. Disclosures Fan: IsoPlexis: Current Employment, Current equity holder in private company; Singleron Biotechnologies: Current Employment, Current equity holder in private company. Levine:Morphosys: Consultancy; Prelude Therapeutics: Research Funding; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Amgen: Honoraria; Astellas: Consultancy; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy. Hoffman:Protagonist: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Dompe: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Forbius: Consultancy.

AbstractActivation of the P53 pathway through inhibition of MDM2 using nutlins has shown clinical promise in the treatment of solid tumors and hematologic malignancies. There is concern, however, that nutlin therapy might stimulate the emergence or expansion of TP53-mutated subclones. We recently published the results of a phase 1 trial of idasanutlin in patients with polycythemia vera (PV) that revealed tolerability and clinical activity. Here, we present data indicating that idasanutlin therapy is associated with expansion of TP53 mutant subclones. End-of-study sequencing of patients found that 5 patients in this trial harbored 12 TP53 mutations; however, only 1 patient had been previously identified as having a TP53 mutation at baseline. To identify the origin of these mutations, further analysis of raw sequencing data of baseline samples was performed and revealed that a subset of these mutations was present at baseline and expanded during treatment with idasanutlin. Follow-up samples were obtained from 4 of 5 patients in this cohort, and we observed that after cessation of idasanutlin, the variant allele frequency (VAF) of 8 of 9 TP53 mutations decreased. Furthermore, disease progression to myelofibrosis or myeloproliferative neoplasm blast phase was not observed in any of these patients after 19- to 32-month observation. These data suggest that idasanutlin treatment may promote transient TP53 mutant clonal expansion. A larger study geared toward high-resolution detection of low VAF mutations is required to explore whether patients acquire de novo TP53 mutations after idasanutlin therapy.

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.

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.

Abstract A limited number of drugs are available to treat patients with polycythemia vera (PV) and essential thrombocythemia (ET). We attempted to identify alternative agents that may target abnormalities within malignant hematopoietic stem (HSCs) and progenitor cells (HPCs). Previously, MDM2 protein levels were shown to be upregulated in PV/ET CD34+ cells, and exposure to a nutlin, an MDM2 antagonist, induced activation of the TP53 pathway and selective depletion of PV HPCs/HSCs. This anticlonal activity was mediated by upregulation of p53 and potentiated by the addition of interferon-α2a (IFN-α2a). Therefore, we performed an investigator-initiated phase 1 trial of the oral MDM2 antagonist idasanutlin (RG7388; Roche) in patients with high-risk PV/ET for whom at least 1 prior therapy had failed. Patients not attaining at least a partial response by European LeukemiaNet criteria after 6 cycles were then allowed to receive combination therapy with low-dose pegylated IFN-α2a. Thirteen patients with JAK2 V617F+ PV/ET were enrolled, and 12 (PV, n = 11; ET, n = 1) were treated with idasanutlin at 100 and 150 mg daily, respectively, for 5 consecutive days of a 28-day cycle. Idasanutlin was well tolerated; no dose-limiting toxicity was observed, but low-grade gastrointestinal toxicity was common. Overall response rate after 6 cycles was 58% (7 of 12) with idasanutlin monotherapy and 50% (2 of 4) with combination therapy. Median duration of response was 16.8 months (range, 3.5-26.7). Hematologic, symptomatic, pathologic, and molecular responses were observed. These data indicate that idasanutlin is a promising novel agent for PV; it is currently being evaluated in a global phase 2 trial. This trial was registered at www.clinicaltrials.gov as #NCT02407080.

Abstract The Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and the prefibrotic form of primary myelofibrosis (PMF), frequently progress to more overt forms of MF and a type of acute leukemia termed MPN-accelerated phase/blast phase (MPN-AP/BP). Recent evidence indicates that dysregulation of the tumor suppressor tumor protein p53 (TP53) commonly occurs in the MPNs. The proteins MDM2 and MDM4 alter the cellular levels of TP53. We investigated in 1,294 patients whether abnormalities involving chromosomes 1 and 12, which harbor the genes for MDM4 and MDM2, respectively, and chromosome 17, where the gene for TP53 is located, are associated with MPN disease progression. Gain of 1q occurred not only in individuals with MPN-BP but also in patients with PV and ET, who, with further follow-up, eventually evolve to either MF and/or MPN-BP. These gains of 1q were most prevalent in patients with a history of PV and those who possessed the JAK2V617F driver mutation. The gains of 1q were accompanied by increased transcript levels of MDM4. In contrast, 12q chromosomal abnormalities were exclusively detected in patients who presented with MF or MPN-BP, but were not accompanied by further increases in MDM2/MDM4 transcript levels. Furthermore, all patients with a loss of 17p13, which leads to a deletion of TP53, had either MF or MPN-AP/BP. These findings suggest that gain of 1q, as well as deletions of 17p, are associated with perturbations of the TP53 pathway, which contribute to MPN disease progression.