ALBERT

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: 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: 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.

Description: Although the clinical course of patients with myelofibrosis (MF) is punctuated by the development of extramedullary hematopoiesis leading to symptomatic splenomegaly, the spleen is not a hematopoietic organ in normal adults. Spleens from patients with MF are characterized by increased numbers of hematopoietic cells and increased micro vessel density within the red pulp. Previously, we reported that the siderophore, lipocalin-2 (LCN2), was elaborated by MF myeloid cells which leads to increased MF marrow fibrosis, osteoblastogenesis,and depletion of the pool of normal hematopoietic cells but promotion of the proliferation of MF cells (Lu M, Blood, 2015). Using nanostring nCounter technology, we have now demonstrated that LCN2 transcript levels were significantly higher in MF spleen (4 fold) than in normal spleen samples (p=0.008) and that LCN2 transcript levels were only modestly increased in MF marrow (p=0.48). The data identified the spleen as a major source of LCN2 in MF patients. These findings led us to further examine the effects of LCN2 on MF spleen CD34+ cells and micro environmental cells. Normal spleen adherent cells (AC) were treated with LCN2 and conditioned media (CM) were collected. CD34+ cells isolated from normal BM or MF spleen were cultured with this CM for an additional 5 days. LCN2 treated AC CM increased MF hematopoietic cell numbers by 2 fold and increased MF CFU-GM and BFU-E numbers by 40% (p

Description: Key Points LCN2 acts to generate reactive oxygen species, leading to increased DNA strand breaks and apoptosis in normal CD34+ cells. LCN2 promotes the generation of osteoblasts but diminishes adipogenesis, resembling the composition of the MF marrow microenvironment.

Description: Objective The marrows and spleens of myelofibrosis (MF) patients are characterized by megakaryocytic (MK) hyperplasia as well as microenvironment (MicroE) abnormalities including increased micro-vessel density, stromal cell (SC) hyperplasia and fibrosis. MF is accompanied by dysregulation of inflammatory cytokines (INF-CyKs) which alter the tissue specific MicroE niches in which MF HSC reside. We hypothesized that MF MKs play a critical role in MF pathobiology and examined their potential to affect MF and normal CD34+ cells as well as marrow and splenic endothelial cells (EC) and SCs. Methods & Results MF MKP/MKs elaborate specific INF-CyKs. CD34+ cells were cultured in serum free medium with stem cell factor and thrombopoietin (TPO) for 7 days, and then cultured with TPO alone for an additional 7 days to generate cell population enriched for MK progenitor cells (MKPs) (CD34+CD41+) and MKs (CD34-/CD41+) (MKP/MK: 22-61% in normal and 27.6-54% in MF). MF MKP/MKs as compared to normal MKPs (nMKP/MKs) contained increased transcripts for several INF-CyKs including: IL-8 (31 fold), TGF-β (8 fold) and VEGF (93 fold). The transcripts for the P53 antagonist MDM2 (112 fold) and the activity of the transcription factor NF-κB were also increased. Furthermore, media conditioned by MF MKP/MKs contained increased protein levels of IL-8, TGF-β and VEGF (5.8, 1.4 and 5.2 fold, respectively) as compared to nMKP/MKs. Using immunohistochemistry, we demonstrated that IL-8 protein was present in normal and MF splenic SCs and ECs, but was exclusively present in MF splenic MKs. Plasma IL-8 levels were significantly elevated in MF patient plasma (p=0.0008). These data indicate that MF MKP/MKs elaborate a series of INF-CyKs which promote the development of MF. IL-8 promotes MF CD34+ cells proliferation. CXCR1 and CXCR2 are two receptors that bind IL-8. Flow cytometric analyses showed that MF CD34+ cells expressed higher levels of CXCR1 and CXCR2 than normal CD34+ cells (3.3 and 3.1 fold, respectively). Addition of IL-8 increased MF CD34+ cell numbers by 2 fold and assayable CFU-GM by 40% (p=0.033), but this effect was eliminated by the addition of reparixin, a CXCR1/CXCR2 antagonist (p=0.0055). MF MKP/MKs can alter the HSC MicroE. Using IHC and flow cytometry we observed that CXCR1 and CXCR2 were also expressed by splenic SCs and ECs and marrow mesenchymal stem cells. When SCs and ECs were incubated with equal numbers of normal or MF MKP/MKs, higher levels of SCF and VEGF were elaborated by SCs but not ECs. This effect was more pronounced with MF MKP/MKs as compared to nMKP/MKs (p=0.02 for SCF and p=0.003 for VEGF). By contrast, higher levels of IL-8 were elaborated by both ECs and SCs following co-cultivation with MF MKP/MKs (p=0.047 and p=0.03). The addition of reparixin to these co-cultures decreased the levels of VEGF and IL-8 to baseline. Co-culturing MF CD34+ cells with either SCs or ECs significantly increased the numbers of CD34+ cells, an effect which could be blocked by the addition of reparixin. These data indicate that MF MKP/MKs provide inflammatory signals that alter the MF MicroE which supports MF CD34+ cells and that these signals can be disrupted by drugs blocking IL-8-CXCR1/2 interactions. MF MKP/MKs and CD34+ cells can be targeted with ruxolitinib, the nutlin-RG7112 and a BET inhibitor. Treatment of MF CD34+ cells with low doses of RG7112, ruxolitinib, or JQ1 alone or in combinations decreased MF but not normal hematopoietic colony formation. Treatment of MF MKP/MKs with each of these agents decreased phosphor-NF-kB p65 levels as demonstrated by western blot and decreased the elaboration of IL-8 by 20 to 60%. Conclusion These data indicate that MF MKPs are characterized by increased transcripts for MDM2 as well as NF-κB activity contributing / leading to the MK hyperplasia characteristic of MF and the elaboration of a group of lineage specific cytokines (TGF-β, IL-8, VEGF) that not only affect MF CD34+ cells but also cells belonging to the MicroE (ECs and SCs) (Figure 1). The MF promoting activities of MF MKP/MKs can be effectively targeted with ruxolitinib, RG7112, and BET inhibitors. Furthermore reparixin can be utilized to interrupt the interactions between IL-8 and CXCR1/2 expressing cells (CD34+ cells, ECs and SCs). These data provide the preclinical rationale for the evaluation of combinations of drugs which can dampen the cascade of events that result when MF MKP/MKs interact with CD34+ cells and MicroE cells. Disclosures Hoffman: Summer Road: Research Funding; Merus: Research Funding; Janssen: Research Funding; Incyte: Research Funding; Formation Biologics: Research Funding.