PHF6 Somatic Mutations and Their Functional Role in the Pathophysiology of Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Abstract Recurrent somatic nonsense PHF6 mutations have been reported in patients with T-acute lymphocytic leukemia, AML and chronic myeloid leukemia in blast crisis. Germ line (GL) PHF6 mutations are responsible for Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation and dysmorphic features. PHF6 is a highly conserved 41kDa protein with ubiquitous expression in hematopoietic cells, including CD34+ cells. We screened patients (N=1166) with myeloid neoplasms by targeted multi-amplicon deep NGS targeting all ORFs of PHF6 to determine the prevalence and distribution and molecular context of PHF6 gene alterations. In total, we identified and verified 52 cases with somatic PHF6 mutations, 32 of which were frameshift or nonsense mutations and with a strong male predominance (76%). Mutations were distributed almost equally between 2 DNA binding domains. Previously, PHF6 has been included in other screening panels (Haferlach et al. 2014 and Papaemmanuil 2013) with somatic mutations found in 24/944 and 21/738 MDS cases, respectively. SNP-array karyotyping showed that microdeletions involving the PHF6 locus were present in about 1.2% of myeloid neoplasms, but affected only female patients. The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (P=.018). The most commonly associated somatic mutations included RUNX1 (P=.001) and IDH1 (P=.008) but not IDH2 (P>.1). There was no impact on overall survival with respect to PHF6 mutant status in total or within individual risk groups (low risk (RA,RARS) vs. high-risk (RAEB1/2). Concomitant PHF6 and RUNX1 mutations were associated with particularly poor prognosis. RUNX1 mutational status correlated with PHF6 expression levels and PHF6 expression inversely correlated with RUNX1 mRNA levels. Subsequent analysis of clonal architecture using VAF calculations and serial samples for these cases suggested that PHF6 may function as a founder driver gene in 18% of cases. PHF6 variant allelic frequency (VAF) varied between disease subtypes, with the highest clonal burden found in AML patients (P<.01). Within MDS patients we also found lower expression of PHF6 mRNA in CD34+ cells in MDS overall vs. controls (P<.01), as well as lower expression of PHF6 in advanced myeloid neoplasms (P<.05). Lower expression (defined as mean+1SD of controls) was found in 12% and 23% of patients with lower- or higher- risk MDS, respectively. Recent studies have proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at G2/M phase and DNA damage. Following shRNA knockdown, hematopoietic cell lines showed only moderately accelerated growth and increased response to growth factors, while EPO-dependent UT7, did not result in growth factor autonomy. To delineate the possible pathophysiological pathway involving PHF6, we compared transcriptional expression profiles of 5 different cell lines with shPHF6 to WT counterparts. We then studied the consequences of PHF6 knockdown on transcriptional profiles. We have found 1020 transcripts differentially expressed (with at least 1.5x change up/down) in the context of shPHF6 knock down. Concordant results among all 5 cell lines resulted in 354 genes that were upregulated and 766 that were down-regulated. Analyses with primary patient data derived from low PHF6 expressors and mutant cases found a concordance of 71 upregulated genes and 80 genes that were downregulated. The most significant functional group of transcripts that was found to be modulated was a family belonging to ribosomal biogenesis pathway (pFDR<1x10-6). Mass spec fingerprinting found protein-protein interaction partners that were found to be dysregulated on a transcriptional level. This finding of protein interaction/transcriptional dependence might suggest feedback mechanisms on a transcriptional level. In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms, frequently associated with RUNX1/IDH1 mutations. Our functional in vitro studies along with recently published reports suggest an association of PHF6 deficiency with transcriptional regulation and thereby provide a basis for a phenotype conveyed by ancestral lesions, consistent with its role as a tumor suppressor gene. Disclosures Sekeres: Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Makishima:The Yasuda Medical Foundation: Research Funding.

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Association of MHC Class I Chain-Related Gene a (MICA) Polymorphisms with Allogeneic Hematopoietic Cell Transplantation Outcomes in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-112745 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2075-2075

Author(s):

Sagar S. Patel ◽

Betty K. Hamilton ◽

Lisa Rybicki ◽

Dawn Thomas ◽

Arden Emrick ◽

...

Keyword(s):

Board Of Directors ◽

Binding Affinity ◽

Myeloid Leukemia ◽

Research Funding ◽

Somatic Mutations ◽

Hematopoietic Cell Transplantation ◽

Advisory Committees ◽

Post Transplant ◽

Transplant Outcomes ◽

Nkg2d Receptor

Abstract Background MHC class I chain-related gene A (MICA) is a polymorphic ligand of the natural killer (NKG2D) receptor on immune effector cells. The activating NKG2D receptor controls immune responses by regulating NK cells, NKT cells and γδ-T cells. Dimorphisms at sequence position 129 of the MICA gene confers varying levels of binding affinity to NKG2D receptor. MICA previously has been associated with post-allogeneic hematopoietic cell transplantation (alloHCT) outcomes including graft-versus-host-disease (GvHD), infection, and relapse. However, it is unclear how MICA interacts with cytogenetic and somatic mutations in regards to these outcomes in acute myeloid leukemia (AML). Methods We conducted a single center, retrospective analysis of adult AML patients in first or second complete remission (CR1, CR2), who underwent T-cell replete matched related or unrelated donor alloHCT. Analysis was limited to those who had MICA data available for donors and recipients. In addition to cytogenetic risk group stratification by European LeukemiaNet criteria (Döhner H, et al, Blood 2016), a subset of patients had a 36-gene somatic mutation panel assessed prior to alloHCT by next-generation sequencing. Dimorphisms at the MICA-129 position have previously been categorized as weaker (valine/valine: V/V), heterozygous (methionine/valine: M/V), or stronger (methionine/methionine: M/M) receptor binding affinity. Fine and Gray or Cox regression was used to identify the association of MICA and outcomes with results as hazard ratios (HR) and 95% confidence intervals (CI). Results From 2000 - 2017, 131 AML patients were identified meeting inclusion criteria. Median age at transplant was 54 years (18-74), with 98% Caucasian. Disease status at transplant included 78% CR1 and 22% CR2. Cytogenetic risk stratification showed 13% of patients as favorable, 56% as intermediate, and 31% as adverse-risk. The five most common somatic mutations were FLT3 (15%), NPM1 (14%), DNMT3A (11%), TET2 (7%), and NRAS (6%). 60% of patients had a related donor. A myeloablative transplant was performed in 84% of patients and 53% had a bone marrow graft source. The most common conditioning regimen used was busulfan/cyclophosphamide (52%). 12% of patients were MICA mismatched with their donor. The distribution of donor MICA-129 polymorphisms were 41% V/V, 53% M/V, and 6% M/M. In univariable analysis, donor-recipient MICA mismatch tended to be associated with a lower risk of infection (HR 0.49, CI 0.23-1.02, P=0.06) and grade 2-4 acute GvHD (HR 0.25, CI 0.06-1.04, P=0.06) but was not associated with other post-transplant outcomes. In multivariable analysis, donor MICA-129 V/V was associated with a higher risk of non-relapse mortality (NRM) (HR 2.02, CI 1.01-4.05, P=0.047) (Figure 1) along with increasing patient age at transplant (HR 1.46, CI 1.10-1.93, p=0.008) and the presence of a TET2 mutation (HR 6.00, CI 1.77-20.3, P=0.004). There were no differences between the V/V and the M/V+M/M cohorts regarding somatic mutational status, cytogenetics and other pre-transplant characteristics and post-transplant outcomes. With a median follow-up of 65 months for both cohorts, 45% vs. 49% of patients remain alive, respectively. The most common causes of death between the V/V and the M/V+M/M cohorts was relapse (38% vs. 62%) and infection (31% vs. 8%), respectively. Conclusion While previous studies have demonstrated associations of somatic mutations and cytogenetics with survival outcomes after alloHCT for AML, we observed mutations in TET2 and the V/V donor MICA-129 polymorphism to be independently prognostic for NRM. Mechanistic studies may be considered to assess for possible interactions of TET2 mutations with NK cell alloreactivity. The weaker binding affinity to the NKG2D receptor by the V/V phenotype may diminish immune responses against pathogens that subsequently contribute to higher NRM. These observations may have implications for enhancing patient risk stratification prior to transplant and optimizing donor selection. Future investigation with larger cohorts interrogating pre-transplant AML somatic mutations with MICA polymorphisms on post-transplant outcomes may further elucidate which subsets of patients may benefit most from transplant. Disclosures Nazha: MEI: Consultancy. Mukherjee:Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; BioPharm Communications: Consultancy; Bristol Myers Squib: Honoraria, Speakers Bureau; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; LEK Consulting: Consultancy, Honoraria; Aplastic Anemia & MDS International Foundation in Joint Partnership with Cleveland Clinic Taussig Cancer Institute: Honoraria. Advani:Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Glycomimetics: Consultancy; Novartis: Consultancy. Carraway:Novartis: Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Speakers Bureau; FibroGen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Speakers Bureau. Gerds:Apexx Oncology: Consultancy; Celgene: Consultancy; Incyte: Consultancy; CTI Biopharma: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: 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. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy. Majhail:Incyte: Honoraria; Anthem, Inc.: Consultancy; Atara: Honoraria.

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The Application of Machine Learning to Improve the Subclassification and Prognostication of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-139013 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-28

Author(s):

Hassan Awada ◽

Arda Durmaz ◽

Carmel Gurnari ◽

Ashwin Kishtagari ◽

Manja Meggendorfer ◽

...

Keyword(s):

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Somatic Mutations ◽

Cross Validation ◽

Steering Committee ◽

Advisory Board ◽

Advisory Committees ◽

Genomic Features ◽

Acute Myeloid

Genetic mutations (somatic or germline), cytogenetic abnormalities and their combinations contribute to the heterogeneity of acute myeloid leukemia (AML) phenotypes. To date, prototypic founder lesions [e.g., t(8;21), inv(16), t(15;17)] define only a fraction of AML subgroups with specific prognoses. Indeed, in a larger proportion of AML patients, somatic mutations or cytogenetic abnormalities potentially serve as driver lesions in combination with numerous acquired secondary hits. However, their combinatorial complexity can preclude the resolution of distinct genomic classifications and overlap across classical pathomorphologic AML subtypes, including de novo/primary (pAML) and secondary AML (sAML) evolving from an antecedent myeloid neoplasm (MN). These prognostically discrete AML subtypes are themselves nonspecific due to variable understanding of their pathogenetic links, especially in cases without overt dysplasia. Without dysplasia, reliance is mainly on anamnestic clinical information that might be unavailable or cannot be correctly assigned due to a short prodromal history of antecedent MN. We explored the potential of genomic markers to sub-classify AML objectively and provide unbiased personalized prognostication, irrespective of the clinicopathological information, and thus become a standard in AML assessment. We collected and analyzed genomic data from a multicenter cohort of 6788 AML patients using standard and machine learning (ML) methods. A total of 13,879 somatic mutations were identified and used to predict traditional pathomorphologic AML classifications. Logistic regression modeling (LRM) detected mutations in CEBPA (both monoallelic "CEBPAMo" and biallelic "CEBPABi"), DNMT3A, FLT3ITD, FLT3TKD, GATA2, IDH1, IDH2R140, NRAS, NPM1 and WT1 being enriched in pAML while mutations in ASXL1, RUNX1, SF3B1, SRSF2, U2AF1, -5/del(5q), -7/del(7q), -17/del(17P), del(20q), +8 and complex karyotype being prevalent in sAML. Despite these significant findings, the genomic profiles of pAML vs. sAML identified by LRM resulted in only 74% cross-validation accuracy of the predictive performance when used to re-assign them. Therefore, we applied Bayesian Latent Class Analysis that identified 4 unique genomic clusters of distinct prognoses [low risk (LR), intermediate-low risk (Int-Lo), intermediate-high risk (Int-Hi) and high risk (HR) of poor survival) that were validated by survival analysis. To link each prognostic group to pathogenetic features, we generated a random forest (RF) model that extracted invariant genomic features driving each group and resulted in 97% cross-validation accuracy when used for prognostication. The model's globally most important genomic features, quantified by mean decrease in accuracy, included NPM1MT, RUNX1MT, ASXL1MT, SRSF2MT, TP53MT, -5/del(5q), DNMT3AMT, -17/del(17p), BCOR/L1MT and others. The LR group was characterized by the highest prevalence of normal cytogenetics (88%) and NPM1MT (100%; 86% with VAF>20%) with co-occurring DNMT3AMT (52%), FLT3ITD-MT (27%; 91% with VAF <50%), IDH2R140-MT (16%, while absent IDH2R172-MT), and depletion or absence of ASXL1MT, EZH2MT, RUNX1MT, TP53MT and complex cytogenetics. Int-Lo had a higher percentage of abnormal cytogenetics cases than LR, the highest frequency of CEBPABi-MT (9%), IDH2R172K-MT (4%), FLT3ITD-MT (14%) and FLT3TKD-MT (6%) occurring without NPM1MT, while absence of NPM1MT, ASXL1MT, RUNX1MT and TP53MT. Int-Hi had the highest frequency of ASXL1MT (39%), BCOR/L1MT (16%), DNMT3AMT without NPM1MT (19%), EZH2MT (9%), RUNX1MT (52%), SF3B1MT (7%), SRSF2MT (38%) and U2AF1MT (12%). Finally, HR had the highest prevalence of abnormal cytogenetics (96%), -5/del(5q) (68%), -7del(7q) (35%), -17del(17p) (31%) and the highest odds of complex karyotype (76%) as well as TP53MT (70%). The model was then internally and externally validated using a cohort of 203 AML cases from the MD Anderson Cancer Center. The RF prognostication model and group-specific survival estimates will be available via a web-based open-access resource. In conclusion, the heterogeneity inherent in the genomic changes across nearly 7000 AML patients is too vast for traditional prediction methods. Using newer ML methods, however, we were able to decipher a set of prognostic subgroups predictive of survival, allowing us to move AML into the era of personalized medicine. Disclosures Advani: OBI: Research Funding; Abbvie: Research Funding; Macrogenics: Research Funding; Glycomimetics: Consultancy, Other: Steering committee/ honoraria, Research Funding; Immunogen: Research Funding; Seattle Genetics: Other: Advisory board/ honoraria, Research Funding; Amgen: Consultancy, Other: steering committee/ honoraria, Research Funding; Kite: Other: Advisory board/ honoraria; Pfizer: Honoraria, Research Funding; Novartis: Consultancy, Other: advisory board; Takeda: Research Funding. Ravandi:Abbvie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Orsenix: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Xencor: Consultancy, Honoraria, Research Funding; Macrogenics: Research Funding; BMS: Consultancy, Honoraria, Research Funding. Carraway:Novartis: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC); Stemline: Consultancy, Speakers Bureau; BMS: Consultancy, Other: Research support, Speakers Bureau; Abbvie: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Jazz: Consultancy, Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Kantarjian:Sanofi: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Honoraria, Research Funding; BMS: Research Funding; Abbvie: Honoraria, Research Funding; Aptitute Health: Honoraria; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Jazz: Research Funding; Immunogen: Research Funding; Adaptive biotechnologies: Honoraria; Ascentage: Research Funding; Amgen: Honoraria, Research Funding; BioAscend: Honoraria; Delta Fly: Honoraria; Janssen: Honoraria; Oxford Biomedical: Honoraria. Kadia:Pfizer: Honoraria, Research Funding; Novartis: Honoraria; Cyclacel: Research Funding; Ascentage: Research Funding; Astellas: Research Funding; Cellenkos: Research Funding; JAZZ: Honoraria, Research Funding; Astra Zeneca: Research Funding; Celgene: Research Funding; Incyte: Research Funding; Pulmotec: Research Funding; Abbvie: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Amgen: Research Funding. Sekeres:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda/Millenium: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

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Clinical Impact of Copy Number Variation Revealed By Next Generation Sequencing in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2021-153302 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4463-4463

Author(s):

Kamila Janusz ◽

Ruth Stuckey ◽

Clara Aparicio Pérez ◽

Cristina Bilbao ◽

Inmaculada Fernández Camacho ◽

...

Keyword(s):

Board Of Directors ◽

Copy Number ◽

Myeloid Leukemia ◽

Genetic Material ◽

Median Number ◽

Advisory Committees ◽

Additional Information ◽

Myeloid Neoplasms ◽

Number Variation ◽

Generation Sequencing

Abstract Introduction: Conventional karyotype analysis is one of the most important diagnostic tools to determine the prognosis of acute myeloid leukemia (AML), in which more than 50% of cases are affected. However, the low sensitivity of this technique hampers the detection of small genetic alterations like Copy Number Variation (CNV) that could affect the pathophysiology and prognosis of the disease. Current modern genomic technologies based on next generation sequencing (NGS) are capable to detect CNV at low frequencies. Objective: To analyse CNV of genes related to myeloid neoplasms profile in AML patients at diagnosis and evaluate their connection with the mutational profile, and its possible influence on the clinical-biological phenotype and prognosis of the disease. Materials and methods: The CNV and mutational profile were analysed in samples from 380 AML patients, from PLATAFO-LMA reference centres (IMIBIC, Córdoba and Dr Negrín Las Palmas de Gran Canaria) by NGS, applying a panel of 30 genes (154 regions) related to myeloid neoplasms (Sophia Myeloid Solution®) on Ilumina Myseq platform. Results: NGS detected CNV in at least one gene in 103 AML patients (27.1%). NGS detected 103 gains and 206 losses of genetic material. The median number of genes affected by CNV was 2 (range 1-12). When comparing with conventional karyotype information, CNV provided additional information in 51% of the cases. The chromosomes 7, 11 and 21 were most affected with CNV, occurring in 79 (76.7%), 40 (38.8%) and 36 (35%) patients, respectively. The gains of genetic material occurred more frequently on chromosome 21 in U2AF1 and RUNX1 genes, in 13 patients each. The loss of genetic material in EZH2 and BRAF genes occurred mutually. Interestingly, we observed the tendency that patients with CNV (loss) in NPM1 gene had shorter overall survival compared to cases with NPM1 mutated and without CNV in this gene (1 month vs. 13 months, p = 0.072) (Figure 1). Moreover CNV (loss) in TP53 gene was associated with mutations in this gene, other than deletions (p <0.05). In addition, NGS detected 390 mutations distributed in 29 genes in 103 AML with CNV. The median number of mutations was 3 (range 1-10) (Figure 2). Furthermore, only 2 patients did not have any mutation in genes analysed. The distribution and frequency of genes affected by CNV and by mutations was different (Figure 3). Conclusions: The CNV of genes related to myeloid neoplasms are frequent in AML patients (27.1%) and provides additional information to the conventional karyotype in half of the cases. The loss of NPM1 gene could affect survival of AML patients. The use of NGS with CNV analysis provides important information on copy number alterations that are not detected by the karyotype, which could significantly affect the pathophysiology of AML and with potential clinical impact, especially in patients with normal karyotype. Figure 1 Figure 1. Disclosures Hernández Rivas: Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Honoraria, 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.

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Dissecting Signaling Network Responses to PAK4 Allosteric Modulators (PAMs) in Cell Subsets within Primary Human Acute Myeloid Leukemia Samples

Blood ◽

10.1182/blood.v126.23.3686.3686 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3686-3686

Author(s):

Paul Brent Ferrell ◽

William Senapedis ◽

Alexander Cook ◽

Erkan Baloglu ◽

Yosef Landesman ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Board Of Directors ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

Leukemia Stem Cells ◽

Allosteric Modulators ◽

Advisory Committees ◽

Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults and has a poor outcome with limited treatment options in patients with relapsed or resistant disease. Therapy resistance in AML is likely related to the inadequacy of therapy within leukemia cell subsets, including leukemia stem cells (LSCs). The p21-activated kinase (PAK) family of proteins was shown to be overexpressed in cancer cells and to play a key role in proliferation, survival, and maintenance of cellular structure. The series of orally bioavailable PAK4 allosteric modulators (PAM) have previously been shown to have activity in hematological cancer cell lines, including those derived from acute myeloid leukemia (AML) (Senapedis et al. Blood124, 2208-2208). Understanding how therapies target cellular subsets within primary patient samples could aid drug development by revealing any subset specific drug effects. In this project, we studied the effects of p21-activated kinase 4 (PAK4) modulation in AML samples. PAK4 modulation has been shown to have significant effects on many intracellular signaling pathways, including PI3K/AKT, MAPK/ERK and WNT/β-catenin pathways (Senapedis et al. Blood124, 2208-2208). It is unknown whether PAMs will have similar activity in primary leukemia cells. Likewise, it is currently unclear to what extent PAMs will differentially impact primary cell subsets including leukemia stem cells and non-malignant cell subsets that may be critical to recovery of bone marrow functions. We have previously shown that the single cell biology platform of flow cytometry is well-suited for dissecting clinically relevant signaling network mechanisms in primary human AML (Irish et al. Cell, 118(2):217-28). Methods: Flow cytometry was used to dissect the impact of an orally bioavailable PAM in AML cell lines and primary patient tissue. Cell lines chosen for this study included NRAS mutant KG-1 and Kasumi-1, which carry t(8;21) and express the AML1:ETO fusion protein. Primary AML biopsies were acquired from bone marrow or blood prior to any treatment and patients were identified and consented for this study according to a local Institutional Review Board-approved protocol. AML tissue samples were viably cryopreserved and then assayed ex vivo. Established protocols were used for phospho-specific flow cytometry, fluorescent cell barcoding, and data analysis in Cytobank (Irish et al. Cell, 118(2):217-28, Doxie and Irish, Curr Top Microbiol Immunol. 377:1-21). Results: Differential effects of PAK4 inhibition were observed between cell lines and among cell subsets from AML patient bone marrow. In leukemia cell lines and patient samples, p-ERK and p-S6 showed marked inhibition via PAM, though degree of inhibition varied. In AML patient samples, PAMs blocked signaling responses in p-ERK specifically in AML blasts, but spared normal CD45hi mononuclear cells (0.88 vs. 0.29-fold reduction (arcsinh scale) in p-ERK at 10 nM). Within the AML blast population, CD34+ CD38- and CD34+ CD38+ AML subsets showed similar PAM dose response via p-ERK. Conclusions: Single cell analysis effectively distinguishes effects of PAK4 inhibition via a series of allosteric modulators of PAK4 (PAMs) on leukemia and non-leukemia subsets in the same sample. PAM reduced immediate p-ERK and p-S6 levels in primary leukemia and cell lines. Notably, inhibition in various subsets within human AML was successfully measured by phospho-flow cytometry. Signaling changes in p-ERK were minimal within non-leukemic mature CD45+ mononuclear cells found in primary patient biopsies. Analysis of CD34+ CD38- cells indicates that PAMs could have activity within leukemia stem cells, and, at least, effect the AML progenitors. These findings support further investigation into the mechanism of action and treatment potential of PAMs in AML. Disclosures Senapedis: Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Irish:Novartis: Honoraria; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; InCyte: Research Funding. Savona:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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LAM-003, a Novel Oral Heat Shock Protein 90 Inhibitor for Treatment of Acute Myeloid Leukemia, Including Wild-Type and FMS-like Tyrosine Kinase 3 (FLT3)-Mutant Disease

Blood ◽

10.1182/blood-2019-125770 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2664-2664

Author(s):

Neil Beeharry ◽

Sean Landrette ◽

Jeff Grotzke ◽

Sophia Gayle ◽

Marylens Hernandez ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Myeloid Leukemia ◽

Cell Lymphoma ◽

Heat Shock Protein 90 ◽

Wild Type ◽

Employment Equity ◽

Advisory Committees ◽

Animal Survival ◽

Equity Ownership

Acute myeloid leukemia (AML) remains a disease with high unmet medical need. While most patients respond to initial therapy, few are cured, relapse rates are high, and most patients eventually develop life-threatening complications. FLT3-mutant disease is a particularly aggressive subtype. Recent approval of drugs targeting FLT3-mutant disease have improved short-term outcomes but not all patients respond, and duration of response is limited by secondary mutations that impede FLT3 inhibitor (FLT3i) binding (intrinsic factors) and by secreted stromal factors that activate alternative pro-survival pathways (extrinsic factors). Heat-shock protein 90 (HSP90) is a chaperone protein involved in many cellular processes and inhibition of HSP90 can have pleiotropic effects in targeting cancer cells such as degradation of oncoproteins that drive survival and proteins that mediate protective signaling. Here, we describe the nonclinical activity of LAM-003, an orally bioavailable HSP90 inhibitor (HSP90i) under clinical development for AML. To assess the anti-leukemic activity of LAM-003, we tested a panel of AML cell lines and primary AML samples. LAM-003 inhibited proliferation of both FLT3-mutant and wild-type cell lines, with preferential activity against cells harboring FLT3-ITD (geometric mean FLT3-ITD EC50 = 670 nM [n=8] vs FLT3 WT EC50 = 1400 nM [n=16]). Additionally, we observed that LAM-003 was potent in a subset of the FLT3 WT cells. To explore whether LAM-003 was effective against tumors driven by oncoproteins that are client proteins of HSP90, we focused on AML cells harboring FLT3-ITD. We confirmed that LAM-003 reduced cell surface FLT3-ITD expression and downstream signaling in MV-4-11 and MOLM-13 cells, consistent with HSP90i-mediated degradation of FLT3-ITD. In BA/F3 cells expressing FLT3-ITD with various secondary resistance mutations, we observed that LAM-003 elicited a dose-dependent reduction of FLT3 mutant cell surface expression. Moreover, BA/F3 cells expressing FLT3-ITD and the F691L mutation exhibited the expected resistance to crenolanib, yet LAM-003 retained anti-proliferative activity. Additionally, MOLM-13 cells harboring a FLT3 D835Y mutation demonstrated expected resistance to the FLT3i sorafenib and tandutinib yet remained sensitive to LAM-003. Finally, primary AML blasts harboring a D835 mutation displayed sensitivity to LAM-003 when tested ex vivo. To evaluate the potential of LAM-003 to overcome bone-marrow-stroma-derived resistance, FLT3-ITD AML cell lines (MV-4-11, MOLM-13, MOLM-14) were assayed in unconditioned or stromal-cell-conditioned medium. Conditioned medium dramatically reduced the potency of FLT3i but LAM-003 demonstrated equal potency under both conditions. We also showed that stromal cell co-culture induced FLT3i resistance in MOLM-13 cells whereas LAM-003 retained potent activity. Recognizing that the inherent genetic heterogeneity of AML blasts limits the curative potential of a single drug, we performed in vitro studies to identify drugs that synergize with LAM-003 in 3 FLT3-ITD AML cell lines. Synergy was demonstrated with FLT3i, daunorubicin, azacitidine or cytarabine, with the most robust synergy being observed with venetoclax. Extending the evaluation to AML cells wild type for FLT3 and cell lines from other hematologic indications (multiple myeloma, diffuse large B-cell lymphoma and mantle cell lymphoma), we found that the synergy was not limited to cells harboring FLT3-ITD, but rather correlated with BCL-2 abundance, suggesting a fundamental mechanism of action that depends on BCL-2 family-mediated survival. Mechanistic studies demonstrated that the combination of LAM-003 and venetoclax inhibited AKT-mediated regulation of GSK3B, resulting in MCL-1 degradation. In vivo studies using a MOLM-13 systemic model of FLT3-ITD AML demonstrated that LAM-003 monotherapy significantly improved animal survival and that the combination of LAM-003 and venetoclax significantly prolonged animal survival compared with each single agent. These nonclinical studies demonstrate that LAM-003 exhibits antileukemic activity, overcomes mechanisms of FLT3i resistance and potently synergizes with existing AML drugs. As such, our data provide strong rationale for evaluation of LAM-003 in an ongoing clinical trial in patients with AML (NCT03426605). Disclosures Beeharry: AI Therapeutics: Employment, Equity Ownership. Landrette:AI Therapeutics: Employment. Grotzke:AI Therapeutics: Employment. Gayle:AI Therapeutics: Equity Ownership. Young:AI Therapeutics: Employment, Equity Ownership. Miller:Incuron, Inc.: Consultancy; Cleveland Biolabs, Inc: Employment, Equity Ownership; Calistoga Pharmaceuticals, Inc.: Equity Ownership; AI Therapeutics: Consultancy, Equity Ownership; VelosBio Inc.: Employment, Equity Ownership; Acerta Pharma, Inc.: Equity Ownership. Xu:AI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rothberg:AI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lichenstein:AI Therapeutics: Employment, Equity Ownership.

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BMS-214662 Eliminates Quiescent and Proliferating Acute Myeloid Leukemia Cells through Activation of Protein Kinase Cβ and Enhances the Efficacy of Cytosine Arabinoside

Blood ◽

10.1182/blood.v116.21.2167.2167 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2167-2167

Author(s):

Francesca Pellicano ◽

Amy Sinclair ◽

Alan Hair ◽

Tessa L. Holyoake ◽

Mhairi Copland

Keyword(s):

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Caspase 3 ◽

Bryostatin 1 ◽

Conventional Chemotherapy ◽

Advisory Committees ◽

Viable Cells ◽

Cd34 Cells ◽

Acute Myeloid

Abstract Abstract 2167 Despite major advances in our understanding of the pathophysiology of acute myeloid leukemia (AML), the prognosis remains poor in most patients. While the majority of patients (∼80%) who tolerate induction therapy enter remission, both early and late relapses are a significant problem. It is postulated that, in some cases, these relapses are due to a population of primitive AML stem cells which are not effectively targeted by conventional chemotherapy agents such as cytosine arabinoside (Ara-C) or standard chemotherapy combinations (e.g. DA, ADE or FLAG-IDA). BMS-214662 (Bristol-Myers Squibb), a cytotoxic farnesyltransferase inhibitor (FTI), preferentially kills non-dividing malignant cells (Lee et al, Proc AACR 2001;42:260S) and in a clinical trial as a single agent had anti-leukemia activity in AML (Cortes et al, J Clin Oncology 2005;23:2805). Previous studies have shown that, in chronic myeloid leukemia (CML), BMS-214662 induces mitochondrial apoptosis of primitive progenitor cells via activation of protein kinase Cbeta (PKCβ; Copland et al, Blood 2008;111:2843; Pellicano et al, Blood 2009;114:4186). We therefore assessed the efficacy of BMS-214662, alone and in combination with Ara-C, in patient-derived CD34+ and CD34- AML cells in vitro using a CFSE-based flow cytometry method to track cell division and caspase-3 activity, PARP cleavage, mitochondrial membrane potential (MMP) and measurement of reactive oxygen species (ROS) to assess apoptosis. PKCβ was assessed by Western blotting ± BMS-214662, ± bryostatin-1, a PKCβ modulator. Primary AML cells were cultured for 72 hours in serum free medium supplemented with 5 growth factors (IL-3, IL-6, Flt-3 ligand, G-CSF and SCF). Conditions studied were: (1) no drug control (NDC), (2) BMS-214662 (250nM; IC50 dose), (3) Ara-C 500nM, and (4) BMS-214662 250nm + Ara-C 500nM (BMS-214662+Ara-C). Total viable cells were reduced in all treatment arms compared to NDC. The combination of BMS-214662+Ara-C produced the greatest cytotoxic effect with total viable cells reduced to <20% of NDC after 72 hours (P=0.05). While Ara-C alone did not reduce the number of undivided CFSEmax AML cells compared to NDC, both BMS-214662 alone and BMS-214662+Ara-C resulted in reductions of 47 and 75%, respectively. Furthermore, BMS-214662+Ara-C significantly reduced undivided CFSEmax AML cells compared to either BMS-214662 or Ara-C alone, (P<0.04 and P<0.03, respectively), indicating at least additive activity of this combination. In primary AML cells, BMS-214662 triggered apoptosis via the intrinsic pathway with loss of MMP, generation of ROS, cleavage of PARP and caspase-3 activation. Further experiments demonstrated that PKCβ was up-regulated in a dose-dependent manner in the presence of BMS-214662. Importantly, BMS-214662 at the concentrations effective against AML CD34+ cells did not initiate apoptosis of normal CD34+ hematopoietic cells. Finally, the addition of the PKCβ modulator, bryostatin-1, abrogated the effects of BMS-214662, resulting in reduced activation of caspase-3. These results are interesting as they identify a common mechanism for induction of apoptosis in quiescent CD34+ cells from different myeloid leukemias (all phases of CML and different AML subtypes) which may be unrelated to the FTI activity of this compound. In conclusion, these results indicate that BMS-214662 induces apoptosis of both quiescent and proliferating AML cells via up-regulation of PKCβ and activation of the intrinsic apoptotic pathway. The combination of BMS-214662 with conventional chemotherapy may represent a novel strategy for remission induction therapy in AML, reducing the risk of relapse. Disclosures: Holyoake: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Copland: Novartis Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Antibody-Targeting of IL-3 Receptor-α Increases the Susceptibility of CD34+ CML Progenitors to Dasatinib-Induced Cell Death,

Blood ◽

10.1182/blood.v118.21.3745.3745 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3745-3745

Author(s):

Eva Nievergall ◽

Deborah L. White ◽

Hayley Ramshaw ◽

Angel F. Lopez ◽

Timothy P. Hughes ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Progenitor Cells ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Blast Crisis ◽

Chronic Phase ◽

Advisory Committees ◽

Cd34 Cells

Abstract Abstract 3745 Despite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) in the treatment of chronic myeloid leukemia (CML), Ph+ CD34+ progenitor cells remain detectable even in patients with stable complete cytogenetic response. Over 40% of patients in stable complete molecular remission will develop molecular relapse within 6 months of stopping imatinib. While the exact causes are largely unknown, one of the proposed mechanisms is the protection of leukemic stem and early progenitor cells by the paracrine or autocrine production of cytokines, such as IL-3, GM-CSF and G-CSF, which activate survival pathways that bypass TKI-induced cytocidal effects. In acute myeloid leukemia (AML), the IL-3 receptor α chain (CD123) is recognized as a specific marker for CD34+/CD38− stem cells and therefore is attracting increasing interest as a therapeutic target. However, the function of CD123 in CML remains to date mostly unexplored. The aim of this study is to investigate potential synergy between TKIs and CSL362 (a humanized antibody version of 7G3 against CD123) in targeting CML progenitor and stem cells. CD34+ and CD34+/CD38− cells were isolated from mononuclear cells of newly diagnosed CML chronic phase and blast crisis patients. Flow cytometry studies indicated significantly increased CD123 expression on CD34+/CD38− cells of CML patients in both chronic phase and blast crisis when compared to normal hematopoietic stem cells (p<0.01 and p<0.001 for chronic phase and blast crisis, respectively; Figure A). A functional relevance of increased CD123 expression was demonstrated by IL-3-dependent increase in STAT5 phosphorylation (260.5% of baseline with 20 ng/ml IL-3; n=12; p<0.001) in CML CD34+ cells. Dasatinib inhibits STAT5 phosphorylation by blocking BCR-ABL signaling but only in the absence of IL-3 (62.5% of baseline for dasatinib alone vs. 130.8% for dasatinib + IL-3; n=3; p<0.01). In agreement, IL-3 effectively rescues dasatinib-induced cell death, as evaluated by AnnexinV/7-AAD staining (103.3% vs. 72.45%, n=5; p<0.01) and CFU-GM colony forming assays (69.39% vs. 46.13% relative to no treatment control; n=4; p<0.05). CSL362, in turn, revokes IL-3-mediated STAT5 phosphorylation (37.12% vs. 130.8%; n=3; p<0.001) and cytoprotection (45.05% vs. 69.39% CFC; n=4; p<0.01). In order to further elucidate the role of CSL362, CML CD34+ cells were cultured with increasing concentrations of dasatinib in the presence of IL-3 and CSL362 or BM4 isotype-matched control antibody. Even at very low dasatinib concentrations, CSL362 significantly reduces CML CD34+ colony forming cells (p<0.05; Figure B). Together these results substantiate a relevant role for IL-3-mediated resistance in CML progenitor cells and additionally confirming the ability of CSL362 to effectively bind to CD123 and impede IL-3 function. CSL362 furthermore has been optimized to mediate antibody dependent cell cytotoxicity (ADCC). CSL362 causes specific cell lysis of CML CD34+ progenitor cells in co-culture with allogeneic Natural killer cells as determined by increased lactate dehydrogenase release (ADCC activity of 42.4% ± 8.1%; n=3) and a decrease in the number of CFU-GM colonies by 74.1 % ± 12.2% (n=3). Collectively, our results indicate that a combination of dasatinib and CSL362 inhibits CML progenitor cell survival more effectively in vitro. Therefore, targeting IL-3 receptor α with CSL362 in chronic phase and blast crisis CML patients might provide a novel specific treatment approach aiding the elimination of refractory chronic myeloid leukemic stem and progenitor cells. A: Flow cytometry analysis reveals that CD123 expression is significantly higher in CD34+/CD38− cells of CML patients in chronic phase (CML-CP) and blast crisis (BC-CML) as compared to normal patients (NP), as previously documented for AML patients. ** p<0.01, *** p<0.001 by unpaired, two-tailed Student's t-test. B: In the presence of IL-3, CSL362 significantly reduces the number of colony forming cells. CD34+ cells of de novo CML-CP patients were cultured with dasatinib (0 to 10 nM) +IL-3 (1 ng/ml) ± CSL362 or BM4 (isotype control for CSL362). After 72 hours of culture live cells were plated for CFU-GM assay and colonies were counted after 2 weeks. Mean ± SE of three independent experiments is shown, n=4, p<0.05 by two-way ANOVA. Disclosures: Nievergall: CSL: Research Funding. White:CSL: Research Funding. Lopez:CSL: Research Funding. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hiwase:CSL: Research Funding.

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Proteomic Profile Of CD34+ Cells From Chronic Myeloid Leukemia Patients and From Normal Donors

Blood ◽

10.1182/blood.v122.21.2712.2712 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2712-2712

Author(s):

Maria Rosaria Ricciardi ◽

Valentina Salvestrini ◽

Mattia Forcato ◽

Roberto Licchetta ◽

Simone Mirabilii ◽

...

Keyword(s):

Cell Proliferation ◽

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Striking Similarity ◽

Proteomic Profile ◽

Advisory Committees ◽

Inhibition Of Proliferation ◽

Cd34 Cells

Abstract Chronic Myeloid Leukemia (CML) is a stem cell disease sustained by a rare population of kinetic quiescent cells, frequently resistant to tyrosine kinase inhibitors (TKIs). The Bcr-Abl oncogene and the resulting fusion protein, in fact, activates multiple cross-talking signal transduction pathways (STP), such as RAS/MEK/ERK, PI3K/Akt, Wnt and STAT5, potentially contributing to TKIs drug resistance. Since increasing evidence reports the cooperation of numerous STP in the control of cell proliferation and survival in CML, the aim of this project was to analyze, at the protein level, the expression and activation profile of various STP involved in the mechanisms of cell proliferation and survival of CML CD34+ cells, as compared to different sources of normal CD34+ cells. CD34+ cells were purified by immunomagnetic separation from peripheral blood (PB) of 7 newly diagnosed chronic phase (CP) CML patients and compared to the normal counterpart obtained from normal bone marrow of three healthy donors (NBM) and/or from umbilical cord blood (CB) of three donors. The phosphorylation status of 46 different proteins belonging to numerous STP and the expression of 32 proteins of the apoptotic machinery were assessed by using a customized direct phase proteome profiler antibody array. The resulting dots were visualised using ECL and quantified by densitometric analysis. CP-CML samples were obtained from patients with WBC counts ranging between 41,900 to 421,400; Sokal score resulted intermediate in six patients and low in one. The comparison between the phospho-proteomic profile of CP-CML CD34+ cells and NBM CD34+ cells showed that the former are characterized by: 1) lower phosphorylation of STAT2 (p=0.023), Chk-2 (p=0.036), a serine/threonine-protein kinase required for checkpoint-mediated cell cycle arrest, and tyrosine kinases of the Src family - Lck, Fyn, Src, particularly Yes (p=0.04) - involved in the regulation of growth and cell survival; 2) higher phosphorylation of p53, both at Ser15 (p=0.047) and at Ser46 (p=0.039), p70S6 kinase (p=0.035), RSK (p=0.046), a mediator of mitogens- and stress-induced activation of several transcription factors, and Pyk-2 (p=0.036), a tyrosine kinase involved in cell adhesion and migration. The analysis of the 32 apoptotic proteins revealed that CD34+ cells from CP-CML, compared to CD34+ cells from NBM, are characterized by: 1) lower expression of the catalase (p=0.044), an enzyme that protects cells from the toxic effects of hydrogen peroxide and promotes growth of normal and neoplastic cells including myeloid leukemia cells; 2) higher expression of some members of the heat shock proteins family - HSP60 and HSP70 (p=0.033). We then compared CD34+ cells obtained from CP-CML with the other normal CD34+ cell source represented by the CB. The proteomic profile indicated a remarkable similarity between the CD34+ from CP-CML and those from CB. Accordingly, the two normal CD34+ cells sources showed some differences: in particular, as for CP-CML CD34+ cells, those from CB had significantly lower phosphorylation of STAT2 (p=0.026), of Chk-2 (p=0.014) and higher phosphorylation of p53 at Ser15 (p=0.05), compared to NBM CD34+ cells. In summary, we reported that CD34+ cells from CP-CML are characterized by a proteomic and phospho-proteomic profile that promotes quiescence status through the inhibition of proliferation. A striking similarity was found between CD34+ cells obtained from CP-CML and those from CB. The two normal sources of CD34 displayed differences in the activation status of selected proteins. The presence of these additional and complex changes in the signaling network of CP-CML must be taken into account for the investigation on novel targeted therapies. Disclosures: Castagnetti: Novartis Farma : Consultancy, Honoraria; Bristol Myers Squibb : Consultancy, Honoraria. Rosti:Bristol Myers Squibb : Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis Pharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau.

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Divergent Dynamics of Epigenetic and Genetic Heterogeneity in Relapsed Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.306.306 ◽

2015 ◽

Vol 126 (23) ◽

pp. 306-306 ◽

Cited By ~ 1

Author(s):

Francine E. Garrett-Bakelman ◽

Sheng Li ◽

Stephen S. Chung ◽

Todd Hricik ◽

Rapaport Franck ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Somatic Mutations ◽

Whole Genome ◽

Rna Seq ◽

Advisory Committees ◽

Time Points ◽

Independent Variable ◽

Acute Myeloid

Abstract Acute Myeloid Leukemia (AML) remains a clinical challenge, with most patients dying of relapsed disease. The complete biological basis of relapse remains unclear. Genetic lesions and heterogeneity have been proposed as key drivers of clinical outcome, yet do not fully explain leukemia relapse. Epigenomic dysregulation is a hallmark of newly diagnosed AML. Plasticity is a core property of the epigenome, enabling cells to adapt to stressful conditions, independent of genetic alterations. Hence we asked whether epigenomic plasticity might contribute to AML progression, have functional consequences and be independent of genetic influences in AML (a question that has not been addressed for any tumor type). Methods. We formed an international consortium to collect and profile paired diagnosis and relapse AML specimens. We extracted DNA and RNA from 138 clinically annotated AML patient samples. We obtained matched germline DNA as genetic controls, and fourteen normal CD34+ specimens as DNA methylation and transcriptome controls. We performed methylome sequencing (ERRBS), genomic sequencing (exomes and targeted resequencing) and transcriptomic (RNA-seq) profiling. For a single patient, more intensive multi-layer profiling (whole genome sequencing, ERRBS, RNA-seq and single cell RNA-seq) was performed at five serial time points. We quantified epigenetic allelic heterogeneity (epialleles) using a novel approach that employs entropy equations (MethClone), and validated epiallele composition using orthogonal methods. Some of the major conclusions are: 1) Epigenetic allelic diversity is an independent variable linked to clinical outcome. Statistically significant epiallele shift (ΔS <-90) was detected at thousands of genomic loci (eloci) at diagnosis. High eloci burden correlated (Wilcoxon test) with a shorter relapse free probability in the entire cohort (p = 0.043) and in intermediate-risk patients based on the Medical Research Council (p= 0.016) and European Leukemia Net (p=0.057) criteria. Multivariate analysis using Cox proportional hazards regression model revealed that the epiallele burden was an independent variable correlated with relapse free survival (p = 0.021). 2) Promoter epialleles are linked to hypervariable transcriptional regulation. We observed substantial change in epiallele burden at relapse versus diagnosis. A subset of the eloci localized to gene promoters. High promoter epiallele variance was significantly associated with high transcriptional variance (p<0.001) based on RNA-seq, including genes that were significantly differentially expressed at relapse. Deconvolution of leukemia blast populations using Single Cell RNA-seq confirmed that the presence of promoter epialleles was linked to hypervariable transcriptional states (p<0.001). 3) AML patients can be classified according to epigenetic allele progression at relapse. K-means clustering based on epiallele shift at diagnosis versus relapse distributed patients into three classes: those with reduced, increasing or stable epiallele burden. Strikingly, there was no correlation between epiallele changes and the patterns of genomic evolution. Furthermore, there was no correlation between epiallele patterns acquired with mutations in epigenetic modifiers or other recurrently mutated genes in AML. 4) Epigenetic heterogeneity upon disease relapse is divergent from the genetic landscape. Integrating whole genome sequencing and methylome analysis we observed that a) significant increases in epigenetic heterogeneity precede significant changes in the abundance of somatic mutations; b) whereas a high number of somatic mutations were shared across all time points, epialleles exhibited dominance of distinct and unique eloci at each time point; and c) the variant epiallele frequency decreased earlier in progression than somatic mutation variant allele frequency, suggesting that epigenetic clonal diversification can precede genetic clonal evolution. Summary. Based on our results we propose that epigenetic allele diversity allows populations of leukemia cells to sample transcriptional states more freely thus creating the potential for greater evolutionary fitness. This provides an additional independent mechanism of plasticity that can explain the resilient nature of AML to adapt and survive exposure to chemotherapy drugs, independent of genetic heterogeneity. Disclosures Perl: Actinium Pharmaceuticals: Consultancy; Asana Biosciences: Consultancy; Arog Pharmaceuticals: Consultancy; Ambit/Daichi Sankyo: Consultancy; Astellas US Pharma Inc.: Consultancy. Becker:Millenium: Research Funding. Lewis:Roche: Honoraria, Other: Travel; Amgen: Other: Travel. Levine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

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Genetic or Pharmacological Inhibition of the Polycomb Group Protein BMI1 Impairs Leukemic Transformation Mediated By the CALM-AF10 Fusion Oncoprotein

Blood ◽

10.1182/blood-2018-99-115552 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 762-762

Author(s):

Karina O. Barbosa ◽

Anwesha Ghosh ◽

Anagha Deshpande ◽

Sayantanee Dutta ◽

Scott A. Armstrong ◽

...

Keyword(s):

Cell Cycle ◽

Board Of Directors ◽

Cell Lines ◽

Myeloid Leukemia ◽

Brdu Incorporation ◽

Wild Type ◽

Advisory Committees ◽

Dose Dependent Decrease ◽

Dose Dependent ◽

Acute Myeloid

Abstract A subset of acute myeloid and lymphoid leukemia cases harbor a t(10;11)(p13;q14) translocation resulting in the CALM-AF10 fusion gene. Standard chemotherapeutic strategies are not very effective in treating patients with CALM-AF10 fusions. Hence, there is an urgent need to identify molecular pathways dysregulated in CALM-AF10 positive leukemias which may lay the foundation for novel targeted therapies. The polycomb repressive complex 1 gene BMI1 is consistently overexpressed in CALM-AF10 leukemias. Previous studies have shown that CALM-AF10 leukemias express high levels of BMI1, regardless of whether the leukemias are myeloid or lymphoid. Our analysis of TCGA acute myeloid leukemia (AML) data confirmed that AML cells with AF10-rearrangements displayed significantly higher expression of BMI1 transcripts compared to cells from non AF10-rearranged AML patients. These observations indicate that BMI1 may be directly activated by AF10-fusion oncogenes as suggested by our previous studies. We sought to investigate the role of BMI1 in CALM-AF10 mediated leukemogenesis using murine and human models of CALM-AF10-mediated AML. First, we tested whether BMI1 deficiency can affect CALM-AF10 mediated oncogenic transformation of hematopoietic stem and progenitor cells (HSPCs). Towards this end, we retrovirally transduced fetal liver cells from Bmi1 wild-type, heterozygous or homozygous null mice with the CALM-AF10 fusion oncogene. Upon plating these cells in colony forming unit (CFU) assays, we observed a significant decrease in the colony formation capacity of the CALM-AF10 fusion transduced cells on a Bmi1 deficient background. Next, we performed Cre-recombinase mediated excision of Bmi1 of already transformed CALM-AF10 myeloid leukemia cells (Bmi1 floxed background). Bmi1 deletion led to a significant reduction in the number of total CFUs compared to Bmi1 wild-type cells, with a particularly striking reduction in the number of blast-like colonies. These experiments, using Bmi1 constitutive or conditional knockout-mice, revealed that CALM-AF10 transformed AML cells are dependent on Bmi1. Recently, selective pharmacological BMI1 inhibitors have been developed. We tested the impact of pharmacologic BMI1 inhibition on a panel of CALM-AF10-driven mouse leukemias with the small molecule inhibitor PTC-209. PTC-209 treatment increased gene expression of the known BMI1-repressed targets Cdkn2a (p16) and Cdkn1a (p21) and led to a dose-dependent decrease in cell proliferation. We also observed a marked increase in Annexin V+ cells upon PTC-209 treatment. In addition, cell-cycle analysis using BrdU incorporation assays revealed a significant decrease in cells in the S-phase, demonstrating that PTC-209 treatment leads to growth arrest and apoptosis in CALM-AF10 AML cells. In order to confirm these findings in human AML with CALM-AF10 rearrangements, we treated human CALM-AF10 positive AML cell lines P31, U937 and KPMOTS with PTC-209. Consistent with our results in the murine AML model, we observed a time and dose-dependent decrease in proliferation of these human cell lines upon PTC-209 treatment. Drug treated human cells also showed concomitant cell-cycle arrest and apoptosis induction, coupled with an increase in expression of BMI1 repressed tumor suppressor genes such as CDKN2A and CDKN1A. In summary, our results demonstrate that BMI1 is a bonafide candidate for therapeutic targeting in AML with CALM-AF10 rearrangements and possibly other CALM-AF10 positive leukemias. We are now assessing clinical-grade BMI1 inhibitors for in vivo efficacy in mouse models of CALM-AF10-mediated AML. Disclosures Deshpande: Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees; A2A Pharma: Membership on an entity's Board of Directors or advisory committees.

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