scholarly journals EZH2 Mutations and Impact on Clinical Outcome Analyzed in 1604 Patients with Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1528-1528
Author(s):  
Sebastian Stasik ◽  
Jan Moritz Middeke ◽  
Michael Kramer ◽  
Christoph Rollig ◽  
Alwin Krämer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Employment Equity ◽  
Advisory Committees ◽  
Mutational Status ◽  
Equity Ownership ◽  
Acute Myeloid

Abstract Purpose: The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and key epigenetic regulator involved in transcriptional repression and embryonic development. Loss of EZH2 activity by inactivating mutations is associated with poor prognosis in myeloid malignancies such as MDS. More recently, EZH2 inactivation was shown to induce chemoresistance in acute myeloid leukemia (AML) (Göllner et al., 2017). Data on the frequency and prognostic role of EZH2-mutations in AML are rare and mostly confined to smaller cohorts. To investigate the prevalence and prognostic impact of this alteration in more detail, we analyzed a large cohort of AML patients (n = 1604) for EZH2 mutations. Patients and Methods: All patients analyzed had newly diagnosed AML, were registered in clinical protocols of the Study Alliance Leukemia (SAL) (AML96, AML2003 or AML60+, SORAML) and had available material at diagnosis. Screening for EZH2 mutations and associated alterations was done using Next-Generation Sequencing (NGS) (TruSight Myeloid Sequencing Panel, Illumina) on an Illumina MiSeq-system using bone marrow or peripheral blood. Detection was conducted with a defined cut-off of 5% variant allele frequency (VAF). All samples below the predefined threshold were classified as EZH2 wild type (wt). Patient clinical characteristics and co-mutations were analyzed according to the mutational status. Furthermore, multivariate analysis was used to identify the impact of EZH2 mutations on outcome. Results: EZH2-mutations were found in 63 of 1604 (4%) patients, with a median VAF of 44% (range 6-97%; median coverage 3077x). Mutations were detected within several exons (2-6; 8-12; 14-20) with highest frequencies in exons 17 and 18 (29%). The majority of detected mutations (71% missense and 29% nonsense/frameshift) were single nucleotide variants (SNVs) (87%), followed by small indel mutations. Descriptive statistics of clinical parameters and associated co-mutations revealed significant differences between EZH2-mut and -wt patients. At diagnosis, patients with EZH2 mutations were significantly older (median age 59 yrs) than EZH2-wt patients (median 56 yrs; p=0.044). In addition, significantly fewer EZH2-mut patients (71%) were diagnosed with de novo AML compared to EZH2-wt patients (84%; p=0.036). Accordingly, EZH2-mut patients had a higher rate of secondary acute myeloid leukemia (sAML) (21%), evolving from prior MDS or after prior chemotherapy (tAML) (8%; p=0.036). Also, bone marrow (and blood) blast counts differed between the two groups (EZH2-mut patients had significantly lower BM and PB blast counts; p=0.013). In contrast, no differences were observed for WBC counts, karyotype, ECOG performance status and ELN-2017 risk category compared to EZH2-wt patients. Based on cytogenetics according to the 2017 ELN criteria, 35% of EZH2-mut patients were categorized with favorable risk, 28% had intermediate and 37% adverse risk. No association was seen with -7/7q-. In the group of EZH2-mut AML patients, significantly higher rates of co-mutations were detected in RUNX1 (25%), ASXL1 (22%) and NRAS (25%) compared to EZH2-wt patients (with 10%; 8% and 15%, respectively). Vice versa, concomitant mutations in NPM1 were (non-significantly) more common in EZH2-wt patients (33%) vs EZH2-mut patients (21%). For other frequently mutated genes in AML there was no major difference between EZH2-mut and -wt patients, e.g. FLT3ITD (13%), FLT3TKD (10%) and CEBPA (24%), as well as genes encoding epigenetic modifiers, namely, DNMT3A (21%), IDH1/2 (11/14%), and TET2 (21%). The correlation of EZH2 mutational status with clinical outcomes showed no effect of EZH2 mutations on the rate of complete remission (CR), relapse free survival (RFS) and overall survival (OS) (with a median OS of 18.4 and 17.1 months for EZH2-mut and -wt patients, respectively) in the univariate analyses. Likewise, the multivariate analysis with clinical variable such as age, cytogenetics and WBC using Cox proportional hazard regression, revealed that EZH2 mutations were not an independent risk factor for OS or RFS. Conclusion EZH mutations are recurrent alterations in patients with AML. The association with certain clinical factors and typical mutations such as RUNX1 and ASXL1 points to the fact that these mutations are associated with secondary AML. Our data do not indicate that EZH2 mutations represent an independent prognostic factor. Disclosures Middeke: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Rollig:Bayer: Research Funding; Janssen: Research Funding. Scholl:Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Abbivie: Other: Travel support; Alexion: Other: Travel support; MDS: Other: Travel support; Novartis: Other: Travel support; Deutsche Krebshilfe: Research Funding; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Hochhaus:Pfizer: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Research Funding. Brümmendorf:Janssen: Consultancy; Takeda: Consultancy; Novartis: Consultancy, Research Funding; Merck: Consultancy; Pfizer: Consultancy, Research Funding. Burchert:AOP Orphan: Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Research Funding. Krause:Novartis: Research Funding. Hänel:Amgen: Honoraria; Roche: Honoraria; Takeda: Honoraria; Novartis: Honoraria. Platzbecker:Celgene: Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding; Roche: Research Funding; Johnson & Johnson: Research Funding; Affimed: Research Funding. Serve:Bayer: Research Funding. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

scholarly journals Genetic Dissection of p53 Driven Senescence of Bone Marrow Mesenchymal Cells in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2625-2625
Author(s):  
Rasoul Pourebrahim ◽  
Peter P. Ruvolo ◽  
Steven M. Kornblau ◽  
Carlos E. Bueso-Ramos ◽  
Michael Andreeff
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Bone Marrow Mscs ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Bone Marrow Mesenchymal Cells ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy characterized by bone marrow infiltration of abnormally proliferating leukemic blasts which results in fatal anemia, bleeding and infectious complications due to compromised normal hematopoiesis. Patients with complete remission (CR) but incomplete blood cell count recovery (CRi) have significantly shorter survival compared to CR patients. Although there is a correlation between CRi and minimal residual disease (MRD), the two variables were shown to be independent risk factors for relapse development (1). The mechanism by which AML induces bone marrow failure in patients is largely unknown. Here, we demonstrate that AML derived MSCs highly express p53 and p21 proteins and are more senescent compared to their normal age-matched controls as demonstrated by high β-galactosidase staining (figure 1. A, B&C). Emerging evidence indicates that the aging of endosteal niche cells results in lower reconstitution potential of hematopoietic stem cells (2). To functionally evaluate the effects of AML on bone marrow MSCs, we utilized a murine leukemia model of the AML microenvironment. We transplanted Osx-Cre;mTmG mice with AML cells and compared the senescence of MSCs in normal bone marrow (Figure 1.D) with AML (Figure 1.E). Consistent with our initial findings in human, AML strongly induced senescence of osteoblasts. This suggests that AML suppresses normal hematopoiesis by inducing senescence in the hematopoietic niche. To address the role of p53 signaling in senescence of MSCs we generated a traceable conditional p53 gain/loss model specifically in bone marrow MSCs using Osx-Cre;mTmG; Mdm2fl/+ and Osx-Cre;mTmG;p53fl/fl mice respectively (Figure 1.F). Deletion of p53 in bone marrow MSCs resulted in an increased population of osteoblasts (GFP+) in Osx-Cre;mTmG;p53fl/fl mice in comparison to Osx-Cre;mTmG mice suggesting that p53 loss in osteoblasts inhibits senescence of osteoblasts. In order to evaluate p53 activity after recombination of p53fl alleles in the osteoblasts, we isolated MSCs from bone marrows and analyzed the expression of p21.P21 was significantly down regulated in osteoblasts (GFP+) derived from Osx-Cre;mTmG;p53fl/fl mice whereas its expression in the hematopoietic cells from same tissue (tdTomato+) remained comparable to p53 wild type suggesting that p21 as the master regulator of senescence is regulated by p53 in bone marrow mesenchymal cells. To evaluate the effect of p53 loss in osteoblasts and its impact on hematopoietic cells, we isolated the GFP+ cells (osteoblasts) and RFP + cells (hematopoietic) by FACS. Senescent cells, non-cell autonomously, modulate the bone marrow microenvironment through the senescence-associated secretory phenotype (SASP). We analyzed the expression of fifteen SASP cytokines by QPCR. Deletion of p53 in bone marrow mesenchymal cells strongly abrogated the expression of several SASP cytokines. Interestingly several Notch target genes such as Hey1 and Hey2 were highly induced in MSCs following p53 deletion suggesting a role for Notch signaling in hematopoietic failure following AML induced MSCs senescence. Our data suggest that AML induces senescence of endosteal niche resulting in hematopoietic failure. These findings contribute to our understanding of the role of p53 in leukemia MSCs and could have broad translational significance for the treatment of hematopoietic failure in patients with AML.Chen X, et al. (2015) Relation of clinical response and minimal residual disease and their prognostic impact on outcome in acute myeloid leukemia. J Clin Oncol 33(11):1258-1264.Li J, et al. (2018) Murine hematopoietic stem cell reconstitution potential is maintained by osteopontin during aging. Sci Rep 8(1):2833. Disclosures Andreeff: Astra Zeneca: Research Funding; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Celgene: Consultancy; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SentiBio: Equity Ownership; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Jazz Pharma: Consultancy; Reata: Equity Ownership.

scholarly journals Clinical Characteristics and Outcome in IDH1/2 Mutant AML Patients - Analysis of 3898 Newly Diagnosed Patients with Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1461-1461 ◽  
Author(s):  
Jan Moritz Middeke ◽  
Christoph Rollig ◽  
Michael Kramer ◽  
Alwin Kramer ◽  
Tilman Bochtler ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Normal Karyotype ◽  
Newly Diagnosed ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Acute Myeloid

Abstract Purpose Mutations of the isocitrate dehydrogenase-1 (IDH1) and IDH2 genes are one of the most frequent alterations in acute myeloid leukemia (AML) and can be found in ~20% of patients at diagnosis. Several IDH inhibitors are currently in late stage clinical development with Enasidenib, an IDH2 inhibitor, being recently approved by the FDA. Previous analyses have reported differential impact on response to chemotherapy and outcome, depending on the IDH-mutation type, co-occurring mutations and cytogenetic abnormalities, as well as the variant allele frequency (VAF) of IDH mutations. In order to better understand its prognostic role, we analyzed newly diagnosed AML patients enrolled in prospective trials of the Study Alliance Leukemia (SAL) to investigate the impact of IDH1/2 mutations on outcome. Patients and Methods All AML patients consecutively enrolled into intensive AML treatment protocols of the SAL or into the SAL registry were included in this analysis. Next-generation sequencing (NGS) on an Illumina MiSeq-system was performed to detect IDH1/2 mutations using pre-treatment samples. Overall survival (OS) and response to therapy were analyzed for all patients with intensive treatment and according to the mutational status. Results Overall, samples of 3898 patients were analyzed. The median follow-up was 91 months (95% CI 87.2 - 93.9). Patients' characteristics are shown in Tbl.1. Three-hundred twenty-nine patients (8.4%) had IDH1 mutations and 423 (11%) had IDH2 mutations; both mutations were found in 12 pts, so the overall mutation rate in IDH1 and 2 was 19% (740/3898 patients). Of the IDH1 variants, the most common ones were the R132C found in 143 patients (43%) and R132H in 137 patients (42%). For IDH2, 324 patients had the R140Q (77%) and 80 patients the R172K (19%) variant. According to the two main variants of the more common IDH2 mutations, as reported before, the IDH2 R172K was mutually exclusive with NPM1 and/or FLT3-ITD mutations. Overall, there was a trend for increased OS for patients with IDH2 R172K (26 vs. 15 months) as compared to those with R140Q. Considering only patients with a normal karyotype and no NPM1/FLT3-ITD mutation, these patients (n=27) had a highly significant better OS than patients with IDH2 R140Q (46.3 vs. 13.1 months, p=.012), supporting the findings published by Papaemmanuil et al. (NEJM 2016). In IDH1-mutated patients, we observed statistically significant differences in baseline characteristics between the two most common mutation types, IDH1 R132C and R132H. Patients carrying the R132C mutation were older (62 vs. 55 years, p=.001), had lower WBC (3.6 vs. 21 Gpt/L, p≤.001) and were less likely to have a normal karyotype (43% vs. 66%, p=.002), NPM1 (23% vs. 66%, p=<.001), and FLT3-ITD mutations (8% vs. 27%, p<.001) than those with the R132H variant. In univariate testing, the CR rate was also statistically significant lower in patients with IDH1 R132C (53% vs. 72%, p≤.001), with a median OS of 12.9 months compared to 17.4 months for patients with R132H variant (p=.08). In multivariate analysis including age, WBC, NPM1 and FLT3 status, and ELN risk, the CR rate was significantly lower in patients with the IDH1 R132C variant (p=.038). The median IDH VAF was 38% (range, 0.1 - 58) with no difference according to the different types of mutation. Patients with a VAF > 30% had a significantly higher BM blast count (73% vs 40% for VAF≤5%) and WBC (21.2 Gpt/L vs. 3.7 Gpt/L) at baseline, but there was no clear impact on CR rate or OS found in multivariate analysis. Conclusion In this large cohort of AML patients with IDH1/2 mutations, we found significant and so far not reported differences for one of the two most prominent mutations types of IDH1. The R132C variant was associated with increased age, lower WBC, and lower NPM1 and/or FLT3 co-mutation rate. Further, these patients had lower CR rates and a trend for shorter OS. For IDH2 we were able to reproduce findings on co-mutations and showed a favorable outcome for intensively treated patients with a normal karyotype and no NPM1/FLT3-ITD mutation and the IDH2 R172K variant, providing additional evidence for classification as a separate AML entity. Disclosures Middeke: Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees. Rollig:Bayer: Research Funding; Janssen: Research Funding. Kramer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Daiichi Sankyo: Consultancy. Scholl:Alexion: Other: Travel support; Abbivie: Other: Travel support; Novartis: Other: Travel support; Deutsche Krebshilfe: Research Funding; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; MDS: Other: Travel support; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees. Hochhaus:Incyte: Research Funding; Pfizer: Research Funding; Takeda: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Research Funding. Brümmendorf:Takeda: Consultancy; Pfizer: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Novartis: Consultancy, Research Funding. Burchert:Novartis: Research Funding; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding; Bayer: Research Funding. Krause:Novartis: Research Funding. Hänel:Amgen: Honoraria; Novartis: Honoraria; Roche: Honoraria; Takeda: Honoraria. Platzbecker:Celgene: Research Funding. Mayer:Johnson & Johnson: Research Funding; Roche: Research Funding; Eisai: Research Funding; Affimed: Research Funding; Novartis: Research Funding. Serve:Bayer: Research Funding. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership. Schetelig:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Janssen: Consultancy, Honoraria; Roche: Honoraria; Sanofi: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding. Stoelzel:Neovii: Speakers Bureau.

scholarly journals p53 Mediated Bone Marrow Mesenchymal Stem Cell Expansion Supports Acute Myeloid Leukemia Development

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 523-523
Author(s):  
Rasoul Pourebrahimabadi ◽  
Zoe Alaniz ◽  
Lauren B Ostermann ◽  
Hung Alex Luong ◽  
Rafael Heinz Montoya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Osteoblast Differentiation ◽  
Hematopoietic Cells ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Mdm2 Inhibitor ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is a heterogeneous disease that develops within a complex microenvironment. Reciprocal interactions between the bone marrow mesenchymal stem/stromal cells (BM-MSCs) and AML cells can promote AML progression and resistance to chemotherapy (Jacamo et al., 2014). We have recently reported that BM-MSCs derived from AML patients (n=103) highly express p53 and p21 compared to their normal counterparts (n=73 p&lt;0.0001) (Hematologica, 2018). To assess the function of p53 in BM-MSCs, we generated traceable lineage specific mouse models targeting Mdm2 or Trp53 alleles in MSCs (Osx-Cre;mTmG;p53fl/fl and Osx-Cre;mTmG;Mdm2fl/+) or hematopoietic cells (Vav-Cre;mTmG;p53fl/fl and Vav-Cre;mTmG;Mdm2fl/+). Homozygote deletion of Mdm2 (Osx-Cre;Mdm2fl/fl) resulted in death at birth and displayed skeletal defects as well as lack of intramedullary hematopoiesis. Heterozygote deletion of Mdm2 in MSCs was dispensable for normal hematopoiesis in adult mice, however, resulted in bone marrow failure and thrombocytopenia after irradiation. Homozygote deletion of Mdm2 in hematopoietic cells (Vav-Cre;Mdm2fl/fl) was embryonically lethal but the heterozygotes were radiosensitive. We next sought to examine if p53 levels in BM-MSCs change after cellular stress imposed by AML. We generated a traceable syngeneic AML model using AML-ETO leukemia cells transplanted into Osx-Cre;mTmG mice. We found that p53 was highly induced in BM-MSCs of AML mice, further confirming our findings in primary patient samples. The population of BM-MSCs was significantly increased in bone marrow Osx-Cre;mTmG transplanted with syngeneic AML cells. Tunnel staining of bone marrow samples in this traceable syngeneic AML model showed a block in apoptosis of BM-MSCs suggesting that the expansion of BM-MSCs in AML is partly due to inhibition of apoptosis. As the leukemia progressed the number of Td-Tomato positive cells which represents hematopoietic lineage and endothelial cells were significantly decreased indicating failure of normal hematopoiesis induced by leukemia. SA-β-gal activity was significantly induced in osteoblasts derived from leukemia mice in comparison to normal mice further supporting our observation in human leukemia samples that AML induces senescence of BM-MSCs. To examine the effect of p53 on the senescence associated secretory profile (SASP) of BM-MSCs, we measured fifteen SASP cytokines by qPCR and found significant decrease in Ccl4, Cxcl12, S100a8, Il6 and Il1b upon p53 deletion in BM-MSCs (Osx-Cre;mTmG;p53fl/fl) compared to p53 wildtype mice. To functionally evaluate the effects of p53 in BM-MSCs on AML, we deleted p53 in BM-MSCs (Osx-Cre;mTmG;p53fl/fl) and transplanted them with syngeneic AML-ETO-Turquoise AML cells. Deletion of p53 in BM-MSCs strongly inhibited the expansion of BM-MSCs in AML and resulted in osteoblast differentiation. This suggests that expansion of BM-MSCs in AML is dependent on p53 and that deletion of p53 results in osteoblast differentiation of BM-MSCs. Importantly, deletion of p53 in BM-MSCs significantly increased the survival of AML mice. We further evaluated the effect of a Mdm2 inhibitor, DS-5272, on BM-MSCs in our traceable mouse models. DS-5272 treatment of Osx-cre;Mdm2fl/+ mice resulted in complete loss of normal hematopoietic cells indicating a non-cell autonomous regulation of apoptosis of hematopoietic cells mediated by p53 in BM-MSCs. Loss of p53 in BM-MSCs (Osx-Cre;p53fl/fl) completely rescued hematopoietic failure following Mdm2 inhibitor treatment. In conclusion, we identified p53 activation as a novel mechanism by which BM-MSCs regulate proliferation and apoptosis of hematopoietic cells. This knowledge highlights a new mechanism of hematopoietic failure after AML therapy and informs new therapeutic strategies to eliminate AML. Disclosures Khoury: Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromic: Research Funding. Bueso-Ramos:Incyte: Consultancy. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy. OffLabel Disclosure: Mdm2 inhibitor-DS 5272

scholarly journals Acute Myeloid Leukemia Expands Osteoprogenitor Rich Niche in the Bone Marrow but Resorbs Mature Bone Causing Osteopenia/Osteoporosis in Animal Models

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 86-86 ◽  
Author(s):  
Bin Yuan ◽  
Stanley Ly ◽  
Khoa Nguyen ◽  
Vivien Tran ◽  
Kiersten Maldonado ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Bone Surface ◽  
Advisory Committees ◽  
Osteoprogenitor Cells ◽  
Equity Ownership ◽  
Acute Myeloid ◽  
Mature Bone

Abstract Acute myeloid leukemia (AML) is one of the most aggressive hematological malignancy that originates in the bone marrow (BM). Despite advances in the molecular characterization of AML, factors regulating its progression are still not known. Among several BM niches that support AML growth in the BM, the osteogenic niche has gained attention in recent years owing to its potential role in leukemogenesis. Genetic alterations in osteoprogenitor cells have been shown to induce myeloid leukemia in mouse models. We reported recently that AML cells induce osteogenic differentiation in mesenchymal stromal cells (MSCs) in the BM to facilitate faster AML engraftment in mice (Battula et al., JCI Insight, 2017). However specifics of this osteogenic niche generated by AML are not known. Here we hypothesize that AML expands osteo-progenitor rich niche in the BM, but that the mature bone is reduced. To determine the type of AML-induced osteo-lineage differentiation in the BM, we generated transgenic reporter mice by crossing Osx-CreERt2 mice with Ocn-GFP; ROSA-tdTomato mice. The resulting triple transgenic mice has the genotype of Osx-CreERt2;Ocn-GFP;ROSA-tdTomato. In these mice the tdTomato (red) positive cells represents osteo-lineage cells that originate from Osterix expressing (Osx+) cells, whereas a GFP+ cell represents an osteocalcin-expressing (Ocn+) mature osteoblast. Seven day old triple transgenic mice were injected with tamoxifen to activate Osx-CreERT2 to mark the Osx+ cells with tomato reporter. To investigate the osteogenic cell type that is induced by AML cells in the bone marrow, we implanted murine AML cells with MLL-ENL fusion proteins into Osx-CreERt2;Ocn-GFP;ROSA-tdTomato mice. Three weeks after implantation of AML cells, the femurs and tibia of these mice were dissected and subjected to histological evaluation using fluorescence microscopy. In control BM without AML, the GFP+ (Ocn+) cells were found in the trabecular bone surface as well as the periosteum of the bone, whereas the tdTomato+ (Osx+)cells were found in the marrow and the bone matrix; this suggests that some of the osteocytes originated from tamoxifen-induced Osx+ osteoprogenitor cells. Interestigly, in mice implanted with AML cells, we found a 3-4 fold increase in Osx+ cells in the marrow compared to normal BM (Fig 1A). However, the number of GFP+ cells on the endosteum and trabecular bone surface was reduced, suggesting that AML cells might expand osteoprogenitor cells but not fully differentiated mature osteoblasts. Next, to investigate whether AML cells affect the mature bone, AML PDX cells developed in our laboratory were implanted into NSG mice. The PDX models usually take 12-14 weeks to achieve >90% engraftment in the peripheral blood which provides ample time to observe alterations in bone composition. At this stage, the mice were subjected to computed tomography imaging to measure bone architecture, volume (BV), mineral density (BMD) and bone volume fraction (BVF). Interestingly, we observed large bone cavities close to epiphysis and metaphysis areas in the femur and tibia of mice with AML (Fig 1B). In addtion, BMD and BVF in these mice were reduced by 20-30% compared to control mice without leukemia. To validate the bone resorption in these mice, bone histomorphometric analysis was performed on femurs and tibias from mice with and without AML. Masson-Goldner's Trichrome staining revealed a 5- to 10-fold decrease in the trabecular and cortical bone thickness in AML femurs compared to normal femurs. Moreover, measurements of osteoclast activation by tartrate-resistant acidic phosphatase (TRAP) revealed positive staining for osteoclasts on the endosteal surface and massive bone resorption in AML bone compared to normal bone. Mechanistic studies showed that AML cells inhibit osteoprotegerin (OPG) ~10 fold in MSCs, a factor that inhibits the RNAK ligand which in turn activates osteoclasts that breakdown the bone. In conclusion, our data suggest that bone homeostasis is dysregulated in AML by induction of osteogenic and osteolytic activities simultaneously. AML cells induce an osteoprogenitor niche but also activate osteoclasts resulting in osteopenia/osteoporosis in mouse models. In-depth analysis of bone remodeling in AML patients could result in new insights into the pathobiology of the disease and provide therapeutic avenues for AML. Disclosures Andreeff: Amgen: Consultancy, Research Funding; Oncolyze: Equity Ownership; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Celgene: Consultancy; Astra Zeneca: Research Funding; Jazz Pharma: Consultancy; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SentiBio: Equity Ownership; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership. Battula:United Therapeutics Inc.: Patents & Royalties, Research Funding.

scholarly journals Nascent Transcript and Single Cell Rnaseq Analysis Defines the Mechanism of Action of the LSD1 Inhibitor INCB059872 in Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2509-2509
Author(s):  
Gretchen Johnston ◽  
Haley E. Ramsey ◽  
Kristy Stengel ◽  
Shilpa Sampathi ◽  
Pankaj Acharya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Treatment ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Myeloid Differentiation ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Upregulated Genes

Drugs targeting chromatin-modifying enzymes have entered clinical trials for myeloid malignancies, including INCB059872, a selective irreversible inhibitor of Lysine-Specific Demethylase 1 (LSD1). LSD1 is a component of the CoREST complex, in which it associates with histone deacetylases 1 and 2, the transcriptional co-repressor, mSin3A or mSin3B, and the REST corepressor (RCOR1), so a role in gene expression was expected. While initial studies of LSD1 inhibitors have suggested these compounds may be used to induce differentiation of acute myeloid leukemia, the mechanisms underlying this effect and dose-limiting toxicities are not well understood. Here, we have used precision nuclear run-on sequencing (PROseq) and single-cell RNA-sequencing (scRNAseq) to show that INCB059872 de-represses GFI1/GFI1B-regulated genes to promote a myeloid differentiation gene signature in AML cells while stalling maturation of megakaryocyte progenitor cells. Within 3 days of treatment with INCB059872, the majority of THP-1, which contain an the MLL-translocation, undergo myeloid differentiation. RNAseq analysis indicated that 24h drug treatment upregulated genes involved in hematopoietic cell lineage, which is consistent with the differentiation. In addition, PROseq was used to measure the effects of INCB059872 on nascent transcription at genes and enhancers, as this is one of the best methods to define enhancer activity. In THP-1 cells after 24h treatment, there were 203 genes with at least a 1.5-fold increase in transcription, while there are nearly 1300 enhancers meeting this threshold. Upregulated genes include those associated with myeloid cell differentiation, such as CSF1R and CD86. Given that LSD1 catalyzes the removal of mono- and di-methyl marks from histone H3, we expected that INCB059872 would cause a buildup of histone methylation. Surprisingly, ChIPseq for H3K4me2 and H3K4me1 showed only subtle changes in these marks after 48h drug treatment in THP-1. Only a handful of LSD1i-induced enhancers overlapped with detectable changes in H3K4 methylation. However, our PROseq data is consistent with the increases in H3K27 acetylation seen with OG86 (a compound that disrupts the LSD1:GFI1 interaction) at GFI1 binding sites (PMID: 29590629). Indeed, motif analysis of INCB059872-upregulated enhancers identified the GFI1 recognition sequence as the most highly enriched. Moreover, siRNA inhibition of key components of LSD1-containing chromatin remodeling complexes pinpointed the CoREST complex as mediating the THP-1 myeloid differentiation effects of INCB059872. To investigate on-target thrombocytopenia seen with LSD1 inhibitors in preclinical studies, we analyzed the bone marrow of wild-type mice treated daily with INCB059872 for 0, 4, or 6 days before harvesting and sorting lin-bone marrow cells for scRNA-seq. Notably, one of the most highly upregulated genes in treated cells was Gfi1b. Unsupervised clustering identified 22 clusters, corresponding to unique subpopulations (Fig. 1A). While the distribution of cells into different progenitor populations was mostly unaffected by drug treatment, these data revealed a striking increase in the proportion of cells from treated mice assigned to a megakaryocyte stem/progenitor cluster. Cells within this expanded cluster expressed stem cell markers such as MYCN and PBX1, but also expressed VWF (Fig. 1B). Thus, LSD1 inhibition caused accumulation of megakaryopoiesis-biased stem cells that failed to mature into efficient platelet producers. Finally, we used scRNAseq to analyze bone marrow from an AML patient who responded to treatment with INCB059872 plus azacytidine (AZA). A pre-treatment bone marrow sample was divided into separate cultures to study the effects of INCB059872, AZA, or the combination. Remarkably, unsupervised clustering of patient cells assigned the majority of INCB059872 and combination-treated cells to clusters that were not found in control- or AZA-treated samples. Cells exposed to INCB059872 had upregulated GFI1 and GFI1B, as well as differentiation-related genes that were also observed in AML cell lines. Overall, these data indicate that INCB059872 affects gene expression with kinetics consistent with a loss of CoREST activity to stimulate differentiation of AML blasts, but the inactivation of GFI1/GFI1B impairs megakaryocyte maturation likely explaining thrombocytopenia seen in preclinical models. Disclosures Stubbs: Incyte Corporation: Employment, Equity Ownership. Burn:Incyte: Employment, Equity Ownership. Hiebert:Incyte Corporation: Research Funding. Savona:Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Selvita: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer Ingelheim: Patents & Royalties; AbbVie: Membership on an entity's Board of Directors or advisory committees.

scholarly journals A Novel Proteomic Profiling of the Bone Marrow Microenvironment Reveals Elevated Levels of the Chemokine CCL23 Isoforms in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2709-2709
Author(s):  
Haydar Celik ◽  
Katherine E. Lindblad ◽  
Bogdan Popescu ◽  
Giovanna Fantoni ◽  
Gege Gui ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Serum Samples ◽  
Advisory Committees ◽  
Extracellular Compartment ◽  
Acute Myeloid

The bone marrow (BM) microenvironment is increasingly recognized as an important contributor to acute myeloid leukemia (AML) pathogenesis. However, despite growing interest in characterizing different components and cellular architecture of the BM niche and their biological significance in leukemogenesis, the proteomic constitution of the BM extracellular compartment that distinguishes a leukemic niche from its normal counterpart has not yet been fully described. We therefore performed a quantitative, large-scale proteomic analysis of 1,305 human proteins of the non-cellular compartment of BM (plasma) samples from ten relapsed or refractory AML patients and from ten age- and sex-matched healthy donors (HDs) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). This screen identified a total of 168 differentially abundant proteins, of which 91 were significantly more and 77 proteins significantly less abundant in leukemic BM compared with healthy marrow (FC ≥ 1.5, FDR ≤ 0.05). Comparative analysis of BM plasma and peripheral blood (PB) serum samples from the same AML patients and HDs revealed 65 similarly regulated proteins (37 up-regulated vs. 28 down-regulated) and 1 differently regulated protein between the two compartments. Out of the total 168 proteins, 102 proteins were specifically dysregulated only in the BM compartment. TruSeq Stranded Total RNA-sequencing (Illumina) was also performed using paired-end 75bp sequencing on a HiSeq 3000. RNA was isolated from PAXgene BM RNA tubes (Qiagen) collected in parallel with samples for proteomic analysis. Results of analysis of differentially expressed transcripts only partially overlapped with those candidates identified from our validated proteomic approach, indicating that sequencing of RNA derived from cellular sources of BM may be a suboptimal screening strategy to determine the true proteomic composition of the extracellular compartment of the AML marrow microenvironment. In addition to several previously reported proteins, our proteomics screen discovered numerous aberrantly expressed proteins in leukemic marrow whose role in AML pathogenesis is currently unknown. Using pathway analysis, we identified sets of proteins enriched for specific biological pathways including RAS, ephrin, PDGF, PI3K/AKT, MAPK, Notch, TLR, JAK-STAT, NFκB, Rap1, and Tie2 signaling pathways. A systems biology analysis approach revealed the highly connected network of cytokines and chemokines as the most striking AML-associated proteomic alteration in the BM. We identified IL-8 as a differentially expressed and key central molecule of this network in AML, consistent with recent reports. Importantly, we also identified significantly elevated levels of CKβ8 and CKβ8-1, alternatively spliced isoforms of the myelosuppressive chemokine CCL23 also known as myeloid progenitor inhibitory factor 1 (MPIF-1) or CKβ8, in both leukemic marrow and PB serum samples (Figure 1). Given the critical importance of cytopenias, often disproportional to the degree of leukemic marrow involvement, in the morbidity and mortality of patients with myelodysplastic syndrome (MDS) and AML, we subsequently confirmed this striking finding by performing orthogonal validation in a larger cohort of MDS and AML patients using an ELISA-based immunoassay. This novel finding suggests the possibility that CCL23 may play a role in suppression of normal hematopoiesis in MDS and AML. In support of this hypothesis, we demonstrated in vitro myelosuppressive effects of CCL23 isoforms on colony formation by human CD34+ hematopoietic stem and progenitor cells (HSPCs) in an in vitro colony forming unit assay, resulting in an approximately 2.5-fold decrease in CFU-GM and an evident decrease in CFU-GEMM counts. In summary, our broad and quantitative proteomic dataset of extracellular factors present in leukemic and normal aging bone marrow has already provided novel mechanistic insights into AML pathogenesis and should serve, together with paired RNA-sequencing information, as a useful public resource for the research community. Disclosures Lai: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Speakers Bureau; Astellas: Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Hourigan:SELLAS Life Sciences Group AG: Research Funding; Merck, Sharpe & Dohme: Research Funding.

scholarly journals Targeting Misfolded p53 and p53 Aggregation to Overcome Resistance to Apoptosis in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3786-3786
Author(s):  
Michael Andreeff ◽  
Jianfang Zeng ◽  
Alice Soragni ◽  
Vivian Ruvolo ◽  
Bing Z Carter ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Protein Aggregation ◽  
Board Of Directors ◽  
Protein Misfolding ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Mdm2 Inhibitor ◽  
Acute Myeloid

The function of wild-type (wt) p53 in acute myeloid Leukemia (AML) is suppressed by MDM2, MDM4 and XPO-1 (Andreeff et al, Exp Hematol, 2016). We propose that wt p53 protein misfolding and cytosolic localization are contributing to its inactivation in AML. Immunofluorescence staining with OpalR TSA amplification demonstrated that p53 is localized both in the nucleus and in the cytosol of AML cells with prominent para-nuclear accumulation. We show here that misfolded wt p53 is localized mainly in the cytoplasm of AML cells, similar to what we reported for mutant (mt) p53 previously (Zeng et al, Blood, 2016). p53 misfolding promotes its aggregation which was recently reported as a novel mechanism promoting loss of its anti-tumor functions (Xu et al, Nat Chem Biol, 2011; Soragni et al, Cancer Cell, 2016). A pro-aggregating segment in the p53 DNA binding domain is exposed when p53 is misfolded. We showed that ReACp53, a cell permeable peptide designed to inhibit the aggregation of this segment, induced apoptosis in ovarian cancers bearing mt p53 (Soragni et al, Cancer Cell, 2016). We also reported that wt p53 AML cells responded to ReACp53 treatment (Zeng et al, Blood, 2016). ReACp53 eliminated misfolded p53, promoted its mitochondrial translocation and induced rapid apoptosis, suggesting that cytoplasmic misfolded wt p53 is a novel target in AML. MDM2 promotes p53 degradation, and inhibitors of MDM2 such as Nutlin derivatives are currently in trials for AML. These molecules inhibit p53 proteasomal degradation and result in p53-mediated apoptosis, as we demonstrated pre-clinically and in a Phase I trial of RG7112 in AML (Andreeff et al, Clin Cancer Res, 2015). p53 aggregation is initiated by protein misfolding, and progresses with increasing accumulation of misfolded p53. While p53 degradation is promoted by MDM2, binding of MDM2 to p53 causes p53 to misfold (Sasaki et al, J Biol Chem, 2007). This raises concerns about induction of p53 misfolding and consequent aggregation in tumors treated with MDM2 inhibitors, which could diminish therapeutic efficacy. We observed that levels of total and misfolded p53 and protein aggregation as identified by Proteostat positivity were MDM2 inhibitors dose- and time-dependent in wt p53 AML cells. This supports the hypothesis that MDM2 inhibition can cause not only p53 misfolding but also aggregation. Consequently, we show that adding a p53-aggregation inhibitor such as ReACp53 to an MDM2 inhibitor to limit p53 misfolding and aggregation results in increased cytotoxic activity in wt p53 AML. Co-aggregation of mt p53 with p63/p73 proteins carrying similar pro-aggregating segments has been reported (Xu et al, Nat Chem Biol, 2011). Next, we tested whether coaggregation could be an additional factor sequestering and inactivating wt p53. High levels of ΔNp73α, a tumor-promoting isoform of p73, can antagonize p53 function possibly through hetero-tetramer formation (Coutandin et al, Cell Death Differ, 2009), resulting in chemoresistance (Kazushi et al, Subcell Biochem, 2014). We hypothesize that upregulated ΔNp73α could constrain wt p53 through protein co-aggregation causing inactivation. Increased levels of misfolded p53 and protein aggregation were detected in both ΔNp73α-overexpressing HEK293T and MOLM13 (M13) cells. ΔNp73α-overexpressing M13 cells were resistant to MDM2 inhibitor-induced apoptosis compared to controls but sensitive to ReACp53. Treatment with Nutlin-derivatives (RG7388 or DS3032b) did not alter ΔNp73α levels but caused dose- and time-dependent increases in total and misfolded p53 and protein aggregation. HEK293T and M13 cells overexpressing ΔNp73α had higher levels of misfolded and aggregated p53, which we interpret as ΔNp73α providing a "seed" to accelerate p53 co-aggregation due to MDM2 inhibition. This suggests that ΔNp73α-overexpression conferred resistance to MDM2-mediated apoptosis that could be overcome by inhibition of p53 aggregation. Thus, combination of Nutlin derivatives and ReACp53 treatment exerted enhanced cytotoxicity in both cells lines. In conclusion, our data supports cytoplasmic, misfolded wt p53 as a novel target in AML and offers a rationale to combine therapeutic approaches supplementing MDM2 inhibition with p53 aggregation-targeting molecules to increase effectiveness. The model of wt p53 aggregation and coaggregation induced by MDM2 inhibition may apply to other cancer types. Disclosures Andreeff: Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership. Carter:Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Ishizawa:Daiichi Sankyo: Patents & Royalties: Joint submission with Daiichi Sankyo for a PTC patent titled "Predictive Gene Signature in Acute Myeloid Leukemia for Therapy with the MDM2 Inhibitor DS-3032b," United States, 62/245667, 10/23/2015, Filed.

scholarly journals Molecular Characterization of Acute Myeloid Leukemia Patients with Normal Karyotype

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2809-2809
Author(s):  
Aziz Nazha ◽  
Manja Meggendorfer ◽  
Sudipto Mukherjee ◽  
Wencke Walter ◽  
Stephan Hutter ◽  
...  
Keyword(s):  
Machine Learning ◽  
Acute Myeloid Leukemia ◽  
Risk Stratification ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Normal Karyotype ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Acute Myeloid

Abstract Background Conventional cytogenetic classification remains one of the most important prognostic factors in acute myeloid leukemia (AML). Approximately 50-60 % of patients (pts) with AML have normal karyotype (NK). NK status has traditionally been associated with intermediate risk AML, but actually represents a heterogeneous group with variable outcomes. Adding mutational data such as NPM1, FLT3-ITD, and CEBPa can improve risk stratification for a subset of pts but does not reflect the genomic complexity and mutation interactions that may impact the overall outcome. In this study, we evaluated the association among several mutations and overall survival (OS) in pts with NK AML using unbiased advanced analytics approaches. Method Genomic and clinical data of 2793 primary AML (pAML) pts were analyzed. A panel of 35 genes that are commonly mutated in AML and myeloid malignancies was included. OS was calculated from the time of diagnosis to time of death or last follow up. To study the association of mutations and OS using an unbiased approach, we applied several machine learning algorithms that included random survival forest and recommender system algorithms (machine learning algorithm analogues to Amazon or Netflix recommender systems, in which a customer who buys A and B is likely to buy C; mutations A and B are determined to be likely associated with mutation C). Results Of 2793 pts with pAML, 1352 (48%) had NK and included in the final analysis. The median age of NK pts was 55 years (range, 18-93). Median WBC, hemoglobin, and platelet count at diagnosis were; 21.3 X 109(range, .2-600), 9.1 g/L (range, 2.7-17.6), and 61 X 109 (range, 5-950), respectively. The median number of mutations/sample was 3 (range, 0-7). The most commonly mutated genes were: NPM1 (49%), DNMT3A (37%), FLT3-ITD (24%), CEBPa (19%), TET2 (17%), IDH2 (17%), and RUNX1 (15%). In univariate Cox regression analysis, mutations in NPM1 (HR .81, median OS 58.3 months[m], p= .008), and CEBPa (single mutant, HR, .8, median OS 91.4 m, and double mutant, HR .69, median OS not reached, p < .001, respectively) were all associated with longer OS, while mutations in DNMT3a (HR 1.26, median OS 24 m, p= .003), FLT3-ITD (HR 1.49, median OS 15.2 m, p< .001), TET2 (HR 1.26, median OS 22.3 m, p= .02), RUNX1 (HR 1.36, median OS 22.3 m, p= .003), SRSF2 (HR 1.58, median OS 20.5 m, p< .001), IDH1 (HR 1.29, median OS 20.5 m, p< .001), and ASXL1 (HR 1.89, median OS 15.6 m, p < .001) were associated with shorter OS. The median OS for pts with 0-2 mutations was 59.3 m (95%CI 38.2- 99.1) compared to 34.1m (95%CI 25.3-49.6) for pts with 3-4 mutations and 16.1m (95%CI 12.4- 24.1) for pts with ≥ 5 mutations, p < .001. Association rules identified several combinations of mutations that impacted OS and are summarized in Figure 1. Based on the median OS of each combination, we divided our pt cohort into favorable, intermediate-1, intermediate-2, and unfavorable categories with median OS of 174.9 m (95%CI 79.4-Not reached), 54.8 m (95%CI 28.7-Not reached), 29.2 m (95%CI 22.8-49.6), and 13.8 m (95%CI 12.2-16.1), respectively, p < .001), Figure 1. These findings suggest that the prognostic impact of molecular data on OS in AML pts with NK is limited when using only one or two mutations (in the exception of TP53 mutations which are present in ~ 1% of NK AML pts), Figure 1. For example, pts with NPM1 mutations can have variable OS depending on presence or absence of mutations in other genes. The median OS for pts with NMP1Mut/FLT3-ITDWt/DNMT3AWt(Mut = mutated, Wt = wild type) was 99.1 m compared to 13.4 m for pts with NPM1Mut/FLT3-ITDMut/DNMT3AMut (triple positive), p < .001, Figure 1. Conclusions We developed genomic combinations that can improve the risk stratification of AML pts with NK using unbiased advanced analytic approaches. These combinations can divide patients into 4 risk categories that may aid physicians in treatment decisions. Such approaches account for the impact of individual mutations and the complexity of genomic interactions on outcomes. Figure 1 Figure 1. Disclosures Nazha: MEI: Consultancy. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Carraway:Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Consultancy, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; FibroGen: Consultancy; Jazz: Speakers Bureau; Novartis: Speakers Bureau. Maciejewski:Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals Combining p38MAPK Inhibitors with a Second Targeted Agent Enhances Blockade of Inflammatory Signaling-Mediated Survival in Acute Myeloid Leukemia Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2726-2726
Author(s):  
Stephen E Kurtz ◽  
Christopher A. Eide ◽  
Narain P. Dubey ◽  
Andy Kaempf ◽  
Shannon K. McWeeney ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Single Agent ◽  
Combination Drug ◽  
Advisory Committees ◽  
Ic50 Values ◽  
Equity Ownership ◽  
Acute Myeloid

Abstract Background : Various pro-inflammatory and stress-related stimuli activate p38 mitogen-activated protein kinase (p38MAPK), triggering cascades of cell proliferation, differentiation, and apoptosis signaling. We have previously found that inflammatory cytokines promote growth and survival in primary cells from acute myeloid leukemia (AML) patients. The downstream mediator of inflammatory pathways is p38MAPK, and blocking this regulator with kinase inhibitors abrogates inflammation signaling in AML cells. Methods : We used a functional ex vivo screening assay to identify small-molecule targeted inhibitors and inhibitor combinations demonstrating selective efficacy across broad categories of leukemia. Primary mononuclear cells isolated from leukemia patients (n=408) were plated in the presence of the p38MAPK inhibitor doramapimod or combinations of doramapimod with a second targeted agent that inhibits or effects a non-overlapping biological pathway. A 7-point concentration series was evaluated for both single agent inhibitors and combinations. Leukemia specimens from 408 unique patients were classified into 5 diagnosis subgroups including acute myeloid leukemia (AML; n=206), acute lymphoblastic leukemia (ALL; n=42), chronic lymphocytic leukemia (CLL; n=115), chronic myeloid leukemia (CML; n=16) and myeloproliferative neoplasms/myelodysplastic syndrome (MPN or MDS/MPN; n=29). IC50 and AUC values were derived from probit-based regression for each response curve. Mutational status for FLT3-ITD and NPM1 were compiled from clinical labs or by capillary electrophoresis using a QiaXcel instrument. Disease status was obtained from clinical chart review. For a subset of AML samples, RNAseq analysis was obtained in parallel to drug sensitivity testing. Single and combination drug treatment IC50 and AUC values were compared within groups by Friedman test, across groups by Kruskal-Wallis test, and with continuous variables by Spearman rank correlation. Results : Among diagnostic subgroups tested, AML specimens showed the lowest median IC50 for doramapimod (1.71 uM), with 75 of 206 samples tested (36%) exhibiting IC50 values < 0.5 uM. In contrast, CLL specimens were significantly less sensitive (median IC50: 4.73 uM), with 27 of 115 samples tested (23%) showing IC50< 0.5 uM. For ALL, CML, and MDS/MPN subgroups, median IC50 values were 10, 3.19, and 6.78 uM, respectively; this translated to 17% of ALL, 19% of CML, and 38% of MDS/MPN samples with IC50< 0.5 uM. Doramapimod was also tested in combination with inhibitors of histone deacetylase (panobinostat), cyclin-dependent kinases 4/6 (CDK4/6; palbociclib), bromodomain and extra-terminal (BET) domain (JQ1), and BCL2 (venetoclax). Doramapimod combinations with panobinostat or JQ1 did not show enhanced efficacy compared to either single agent. However, combining doramapimod with palbociclib or venetoclax resulted in significantly enhanced efficacy compared to each single agent (median IC50: 0.014 and 0.075 uM, respectively; p<0.0001). Similar results were obtained using AUC as a drug effect measure. Of the 206 AML samples tested, 75% were sensitive (IC50 values < 0.5 uM) to both of these combinations, whereas 8% and 14% were sensitive to doramapimod in combination with only palbociclib or venetoclax, respectively. Sensitivity to combinations of doramapimod and palbociclib or venetoclax was not significantly associated with either age or gender. For the doramapimod + palbociclib combination, drug sensitivity (AUC) was correlated with gene expression for p38MAPKδ(Spearman r: -0.25). For doramapimod + venetoclax, sensitivity was correlated with expression of BCL2, MCL1, p38MAPKγ (Spearman r: -0.53, 0.23, and -0.33, respectively). Further analysis to align drug sensitivities with additional clinical and genetic features and inflammatory gene expression for AML patient samples is in progress. Conclusions : AML patient specimens demonstrate ex vivo sensitivity to inhibition of p38MAPK, and this efficacy is enhanced when combined with inhibitors of either CDK4/6 or BCL2, suggesting dual inhibition of these pathways may extend clinical utility among patients with genetically heterogeneous leukemia. Disclosures Druker: Celgene: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Millipore: Patents & Royalties; McGraw Hill: Patents & Royalties; Henry Stewart Talks: Patents & Royalties; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; Monojul: Consultancy; ARIAD: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy. Tyner:Genentech: Research Funding; Aptose: Research Funding; Janssen: Research Funding; Array: Research Funding; Incyte: Research Funding; Takeda: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding; Gilead: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees.

CX-01, a Low Anticoagulant Heparin, May Enhance Count Recovery and Treatment Efficacy in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5220-5220 ◽  
Author(s):  
Tibor J. Kovacsovics ◽  
Alice S. Mims ◽  
Mohamed E Salama ◽  
Jeremy M Pantin ◽  
Ken M Kosak ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Treatment Efficacy ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Acute Myeloid ◽  
Count Recovery

Abstract Acute myeloid leukemia (AML) therapy is associated with pancytopenia and with a high failure rate due to resistant leukemia stem cells that home to marrow niches. We present here updated results of this study that combined the low anticoagulant heparin CX-01 with chemotherapy for the treatment of AML. Induction consisted of cytarabine and idarubicin (7+3) along with CX-01. Patients younger than 60 received consolidation therapy with high dose cytarabine along with CX-01. Twelve patients were enrolled (median age 56; range 22 - 74; 3 women). Three, 5, and 4 patients had good, intermediate, and poor risk disease, respectively. Two patients did not finish induction due to events unrelated to CX-01. Day 14 bone marrows were available on 11 patients and were aplastic in all without detectable leukemia. Eleven patients (92%) had a morphologic complete remission after one induction (CR1), including all patients with de novo AML (11/11). No patient required re-induction at Day 14. Eight patients are alive at a median follow-up of 24 months (range 13 - 29). Four patients remain in CR1. Four patients received an allogeneic stem cell transplant in CR1. All patients who received a full induction were evaluable for platelet recovery and had a median day to an untransfused platelet count ≥ 20,000/μl of 21. Five patients who received a full induction without myelopoietic growth factor support were evaluable for neutrophil recovery and had a median time to a neutrophil count ≥ 500/mL of 22 days (mean 22.6 ± 2.4, range 21 - 27). Median disease free survival was 14.8 months. Median overall survival was not attained at the maximum follow up time of 29.4 months. No CX-01-associated serious adverse events occurred. In a supplemental surface plasmon resonance study, CX-01 inhibited binding of CXCL12 to surface conjugated heparin with a 50% inhibitory concentration (IC50) of 4.7 nM, suggesting the possibility that CX-01 may enhance CR by disrupting the CXCL12-mediated leukemic stem cell marrow niche. We conclude that CX-01 is well tolerated when combined with intensive therapy for AML. It appears to be associated with enhanced count recovery and treatment efficacy. Thus, CX-01 warrants further study in this setting. Disclosures Kovacsovics: Seattle Genetics: Research Funding. Deininger:CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kennedy:Cantex Pharmaceuticals: Equity Ownership. Marcus:Cantex Pharmaceuticals: Employment, Equity Ownership. Shami:JSK Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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