NF-κB-Dependent Activation of the Proteasome Components, PSMD1 and PSMD3, As a Mechanism of Resistance to Imatinib

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 are remarkably effective therapies in chronic myeloid leukemia (CML). Despite clinical success, TKIs do not target the CML leukemic stem cell (LSC), and the majority of patients must be treated for life to maintain remission. Our previous work has shown that BCR-ABL1-independent resistance is driven by STAT3 in CML stem/progenitor cells (Eiring et al. Leukemia 2015). Unexpectedly, RNA-sequencing on TKI-resistant K562 cells (K562-R) versus parental controls (K562-S) revealed that resistance is not associated with STAT3-mediated transcription, but is rather reminiscent of TNFa signaling via NF-κB (p=0.024). Nucleocytoplasmic fractionation confirmed these findings, demonstrating higher levels of phospho-NF-κB in the nucleus of CD34+ cells from TKI-resistant patients (n=3) compared to newly diagnosed CML patients (n=2) or normal individuals (n=2). Surprisingly, ELISA results revealed that K562-R cells do not produce autonomous TNFa, but they do produce IL-6 (p<0.01). These data suggest that NF-κB may be driving the gene expression signature of BCR-ABL1-independent resistance, and implicate non-canonical functions for STAT3. To better understand the mechanism by which NF-κB drives resistance, we correlated our RNA sequencing data with gene expression profiles of CML patients not responding to imatinib (McWeeney et al. Blood 2010), identifying 36 genes commonly dysregulated in both TKI-resistant cell lines and patient samples. Of the 30 upregulated genes, 21 had p65-NF-κB bound to their promoter regions via ChIP in hematopoietic cells (UCSC Genome Brower). Two of these genes are members of the ubiquitin proteasome system, including PSMD1 and PSMD3, both of which were implicated as hits in a previously published shRNA library screen for BCR-ABL1-independent resistance (Khorashad et al. Blood 2015). PSMD1 and PSMD3 are non-ATPase subunits of the 19S regulatory complex in the 26S proteasome, likely involved in proteasome substrate recognition and binding. In breast cancer, PSMD1 was shown to regulate cell growth by inducing p53 degradation (Okumura et al. 2018), whereas PSMD3 was shown to protect HER2 from degradation (Fararjeh et al. 2019). qRT-PCR confirmed upregulation of PSMD1 and PSMD3 by 3-fold and 6-fold, respectively, in K562-R cells versus parental controls in the presence of imatinib. Interestingly, according to data from The Cancer Genome Atlas (TGCA), higher levels of PSMD1 and PSMD3 mRNA correlates with a worse prognosis in acute myeloid leukemia (PSMD1, p=0.0138; PSMD3, p=0.0229). We hypothesized that PSMD1 and PSMD3 upregulation contributes to NF-κB activation and TKI resistance. We used doxycycline-inducible shRNAs to assess the function of PSMD1 and PSMD3 in CML cell survival and TKI response. Induction of knockdown (100 ng/mL doxycycline, 72h) resulted in a reduction of PSMD1 and PSMD3 mRNA and protein by ~73% and ~77%, respectively, in K562-R cells. Importantly, immunoblot analysis revealed that knockdown of either PSMD1 or PSMD3 in TKI-resistant K562-R cells resulted in a significant reduction of phospho-NF-κB (p65), suggesting that upregulation of these proteins promotes NF-κB activation. Reduced phospho-NF-κB (p65) correlated with phenotypic effects, including reduced colony formation, increased response to TKIs as assessed in MTS assays, and increased apoptosis in both the presence and absence of imatinib. Our results suggest that NF-κB activation in TKI resistance depends on the proteasome components, PSMD1 and PSMD3, forming a positive feedback loop potentiating NF-κB signaling. Our data also suggest that specific targeting of the ubiquitin proteasome system through either PSMD1 or PSMD3 may be a novel strategy to restore TKI sensitivity in patients with BCR-ABL1-independent TKI resistance. Future studies will address the non-canonical functions of STAT3 in TKI resistance. Disclosures Milojkovic: Novartis: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau. Apperley:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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MLN4924, a Novel Investigational NEDD8 Activating Enzyme Inhibitor, Exhibits Preclinical Activity In Multiple Myeloma and Waldenström's Macroglobulinemia through Mechanism Distinct From Existing Proteasome Inhibitors

Blood ◽

10.1182/blood.v116.21.2988.2988 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2988-2988

Author(s):

Douglas W. McMillin ◽

Zachary Hunter ◽

Jake Delmore ◽

Val Monrose ◽

Peter G Smith ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Normal Donor ◽

Advisory Committees ◽

Safety Studies ◽

Ubiquitin Proteasome ◽

Efficacy Studies

Abstract Abstract 2988 Background: Multiple myeloma (MM) and Waldenström Macroglobulinemia (WM) have both shown clinical responses to Bortezomib therapy which blocks the elimination of ubiquitin tagged regulatory proteins by the proteasome. The NEDD8 activating enzyme (NAE)-inhibitor MLN4924 is a novel agent which demonstrates selective inhibition of the proteins for degradation in the ubiquitin pathway and may offer benefits to MM and WM patients through the more targeted approach. Methods: A panel of human MM and WM cell lines were tested for their in vitro response to MLN4924 using MTT colorimetric survival assays. MM and WM cell lines tested exhibited dose and time dependent decrease of their viability upon exposure to MLN4924 (IC50=25-150 nM). In addition, miRNA and gene expression studies in response to MLN4924 were compared to treatment of the same cells with bortezomib. In vivo safety studies were performed in mice and animal efficacy studies are ongoing in both MM and WM engrafted mice. Results: A panel of MM and WM cells were treated with MLN4924 for 72hrs and compared to the colon carcinoma line HCT116 and normal cell lines HS-5 (stroma) and THLE-3 (hepatocytes). In addition, a longitudinal assessment of viability of MM1S (MM) and BCWM1 (WM) cells during a 72hr incubation with MLN4924 (500nM) showed commitment to death <48hrs. This result, coupled with the observation that normal donor peripheral blood mononuclear cells (PBMCs) and HS-5 stromal cells were less sensitive (IC50 >1000 nM) than the MM or WM cell lines tested, suggest that this compound exhibits a rapid, tumor-selective effect at clinically relevant conditions. We also evaluated primary MM (CD138+) and WM (CD19+) patient bone marrow cells and observed sub-μ M activity by MLN4924. In addition, we tested a series of combinations of MLN4924 with dexamethasone, doxorubicin and bortezomib in both MM1S and BCWM1 cells lines and observed additive activity or greater with MLN4924. Gene expression profiling revealed distinct signatures, in MM1S and BCWM1 lines, as well as distinct patterns of gene expression changes which were induced by MLN4924 vs. bortezomib. For instance, while bortezomib potently induces a compensatory upregulation of transcripts for ubiquitin/proteasome and heat shock protein genes which, in MM1S or BCWM1 cells, were not observed in response to MLN4924 treatment. Additional studies with the proteasome inhibitor MLN9708 revealed similar patterns of expression as bortezomib. These results indicate that MLN4924 does not induce pronounced proteotoxic stress in MM or WM cells, highlighting the distinct effect of MLN4924 on the ubiquitin/proteasome pathway compared to inhibitors which target the 20S proteasome subunit. Longitudinal miRNA profiling revealed a distinct pattern of miRNA expression in MLN4924-treated vs. bortezomib-treated MM and WM cells. Lastly, animal safety studies showed that MLN4924 was tolerated at doses up to 60mg/kg 2x daily for 1 week. Efficacy studies in MM and WM are ongoing. Conclusions: MLN4924 induces cell killing at sub-μ M concentrations for both MM and WM cells with higher sensitivity of tumor cells compared to normal tissues, exhibits selective gene expression and miRNA regulation and can be safely administered to mice. These studies provide the framework for the clinical investigation of MLN4924 in MM and WM. Disclosures: McMillin: Axios Biosciences: Equity Ownership. Smith:Millennium: Employment. Birner:Millennium: Employment. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Treon:Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

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Distinct Gene Expression Patterns of Minimal Residual Disease (MRD) Cells in High-Risk AML Patients Identified By RNA-Sequencing

Blood ◽

10.1182/blood-2018-99-120260 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2757-2757

Author(s):

Christopher B. Benton ◽

Ahmed Al Rawi ◽

Feng Wang ◽

Jianhua Zhang ◽

Jeffrey L. Jorgensen ◽

...

Keyword(s):

Gene Expression ◽

High Risk ◽

Board Of Directors ◽

Rna Sequencing ◽

Research Funding ◽

Mononuclear Cells ◽

Post Treatment ◽

Advisory Committees ◽

Diagnostic Samples ◽

Equity Ownership

Abstract INTRODUCTION Evolving techniques have made possible the direct detection, physical isolation, and study of AML minimal residual disease (MRD) after treatment. This could allow for better identification of therapeutic vulnerabilities in AML. Prior studies have focused on cells that initiate leukemia in mouse models, known as leukemia-initiating cells (LIC), generally with a foundational CD34+CD38- immunophenotype. LIC are typically derived from diagnostic samples of untreated patients. Such stem-like cells do not necessarily represent the residual fraction of AML after treatment. Relapse may originate from non-LIC, and the presence of phenotypically and molecularly defined MRD is now firmly established as a critical prognostic factor for patients. High-risk AML is characterized by relapse, despite morphologic complete remission with initial therapy in most cases. RNA-sequencing was performed on pre- and post-treatment AML subpopulations, including MRD, from high-risk patients, to determine differences in gene expression. METHODS Matched primary AML samples were collected from marrow and peripheral blood of patients with high-risk AML (including patients with unfavorable karyotype and/or TP53 mutation) at diagnosis and after treatment. Mononuclear cells were flow-sorted for bulk (CD45dim) and LIC (Lin-CD34+CD38-CD123+) from diagnostic samples. Post-treatment samples were sorted for bulk mononuclear cells (MNC) and MRD, based on difference-from-normal/MRD immunophenotype specific for each patient as determined from established 20-marker clinical flow cytometry analysis. RNA was isolated using low-input methodology, and RNA-sequencing was performed using Illumina HiSeq 2000. Gene expression was assessed using GO-Elite, and differences between patients and subpopulations were assessed using rank product method. RESULTS Gene expression in MRD was analyzed by RNA-sequencing in comparison to diagnostic samples in eight patients with high-risk AML. Four patients had unfavorable karyotype, including two with TP53 mutations. Patients had additional high-risk features, such as FLT3-ITD or RUNX1 mutations, or secondary/therapy-related AML. Treatment consisted of chemotherapy (6/8) or hypomethylating agents (2/8), with or without other targeted drugs. Residual leukemia was detected in post-treatment samples in all study patients. Significant differences in gene expression were detected between MRD and other sorted populations, including diagnostic bulk AML and LIC. Relevant MRD pathways included those with strong interactions with the microenvironment. Anti-apoptotic mechanisms, cytoskeletal, and cell adhesion related genes, WNT/beta-catenin signaling, and TGFbeta signaling ranked among the most relevant processes in AML MRD subpopulations (Figure 1A, GO-Elite interactome of highly expressed genes in AML MRD). To identify potentially critical and unique MRD-specific genes, rank product method was applied using 1) the most highly expressed genes in AML MRD, 2) the most differential expressed genes between MRD and bulk AML at diagnosis, and 3) the most differentially expressed genes between MRD and bulk MNC after treatment. Among the top 50 scoring genes using this approach (Figure 1B), 16 genes were among the top 5% of genes expressed in MRD among all patients and 20 genes have cell surface gene-products (shown in yellow). Several potential leukemia- and cancer-related genes of interest were identified (shown in bold). CONCLUSIONS Key differences exist between the gene expression profiles of post-treatment MRD from high-risk AML patients, in comparison to other populations and subpopulations of sorted cells before and after treatment. The highlighted differences suggest that MRD relies on specific intrinsic gene expression changes and microenvironmental interactions, and therefore may be targetable after elimination of bulk AML with initial therapy. Accessible surfacesome targets are among top hits. Disclosures Konopleva: cellectis: Research Funding; Immunogen: Research Funding; abbvie: Research Funding; Stemline Therapeutics: Research Funding. Andreeff:Astra Zeneca: Research Funding; Amgen: Consultancy, Research Funding; Jazz Pharma: Consultancy; Celgene: Consultancy; Reata: Equity Ownership; SentiBio: Equity Ownership; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Stroma-Based Activation of pSTAT3Y705 Confers Resistance to FLT3 Inhibitors in FLT3 ITD-Positive AML

Blood ◽

10.1182/blood.v128.22.34.34 ◽

2016 ◽

Vol 128 (22) ◽

pp. 34-34 ◽

Cited By ~ 1

Author(s):

Ami Patel ◽

Anthony D. Pomicter ◽

Anna M. Eiring ◽

Than Hein ◽

William L. Heaton ◽

...

Keyword(s):

Tyrosine Kinase ◽

Board Of Directors ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

Clinical Relapse ◽

Intrinsic Resistance ◽

Advisory Committees ◽

Tki Resistance

Abstract Acute myeloid leukemia (AML) is an aggressive hematopoietic neoplasm that carries the worst prognosis among the hematologic malignancies. Up to 30% of AML patients exhibit activating mutations in FLT3 tyrosine kinase. FLT3 internal tandem duplications (ITDs) comprise ~70% of these mutations and are associated with a poor prognosis. Most patients treated with a single-agent FLT3 tyrosine kinase inhibitor (TKI) relapse within months due to secondary mutations in the FLT3 tyrosine kinase domain (TKD). Results from trials of FLT3 TKIs in AML reveal that leukemic blasts are more easily cleared from peripheral blood than from bone marrow (BM), suggesting that the BM microenvironment promotes survival of AML cells, including leukemia initiating cells, despite inhibition of FLT3. In this conceptual framework, extrinsic factors allow AML cells to survive TKI exposure until AML cell-intrinsic resistance is conferred by FLT3 TKD mutations, leading to clinical relapse. Here, we investigated the role of the BM microenvironment in protection of FLT3+AML cells from treatment with AC220 (quizartinib), a clinically available FLT3 TKI. To investigate the potential of the BM microenvironment to mediate TKI resistance in AML, we cultured FLT3-ITD+ AML cell lines, including MOLM-13, MOLM-14 and MV411, and the CML cell line, K562 (control; FLT3 wild-type), with graded concentrations of AC220 under the following conditions: (i) in regular medium (RM), (ii) in direct contact (DC) with human HS-5 BM stromal cells, or (iii) in HS-5 conditioned medium (CM). Cell proliferation and apoptosis assays revealed that, in RM,AC220 reduced proliferation and increased apoptosis of MOLM-13, MOLM-14 and MV411 cells, but had no effect on K562 cells. DC greatly reduced the effects of AC220 in all three FLT3-ITD+ AML cell lines, with comparable results observed between DC and CM. To confirm these data using primary cells, CD34+ blasts from a patient with newly diagnosed FLT3-ITD+ AML were similarly cultured in RM versus CM ± AC220. Consistent with results in cell lines, CM rescued primary AML cells from AC220-mediated cell death. These data indicate that soluble factors from the BM environment protect FLT3-ITD+ cells from the effects of FLT3 inhibition. Our lab and others have demonstrated that HS-5 DC and CM activate STAT3 in chronic myeloid leukemia, which mediates resistance to BCR-ABL1 TKIs (Bewry et al. Mol Cancer Ther 2008, Traer et al. Leukemia 2012, Eiring et al. Leukemia 2015). To interrogate the role of STAT3 in BM-mediated protection of AML cells from FLT3 inhibition, all cell lines were assessed for pSTAT3Y705 and total STAT3 by immunoblot analysis under each culture condition. In FLT3-ITD+ AML cells grown in RM, pSTAT3Y705 was undetectable, irrespective of AC220 dose. In contrast, pSTAT5Y694 was readily detected at steady state and suppressed by AC220. AML cells cultured in HS-5 DC or in HS-5 CM exhibited strong upregulation of pSTAT3Y705 that was unaffected by AC220, suggesting that soluble factor(s) promote STAT3 activation in AML. pSTAT5Y694, on the other hand, was slightly elevated by HS-5 DC or CM, but remained under control of FLT3 kinase activity. In order to mechanistically implicate STAT3 activation in stroma-based protection, we used a retroviral shRNA construct to knockdown STAT3 (shSTAT3) compared to an empty vector control (LMP) in MOLM-14 cells. STAT3 knockdown (~70%) was confirmed by qRT-PCR and immunoblot analyses. Cells containing shSTAT3 and LMP were cultured for 72 hours in RM or CM ± AC220, followed by analysis using MTS assays. As expected, CM increased the IC50 of AC220 from 1.37 nM to 6.24 nM in LMP-expressing cells (n=3). In contrast, shSTAT3 reduced the IC50 of AC220 from 6.24 nM to 2.87 nM (n=3) in CM, with minimal effects in RM. Similarly, pharmacologic inhibition of STAT3 using the novel STAT3 inhibitor, BP-5-087 (Eiring et al. Leukemia 2015), reduced the IC50 of AC220 from 10.07 nM to 5.91 nM in CM. Analogous experiments in additional FLT3-ITD+cell lines and primary AML cells, using shSTAT3, dominant-negative STAT3 constructs and BP5-087 are ongoing. Our data suggest that STAT3 is a critical signaling node in FLT3-independent TKI resistance mediated by the BM microenvironment. Therapeutic strategies designed to combine FLT3 and STAT3 inhibition may inhibit the survival of leukemic cells in the BM niche, thereby preventing subsequent clinical relapse conferred by TKD mutations. Disclosures Deininger: Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Integrated Genomic, Functional and Prognostic Characterization of Atypical Chronic Myeloid Leukemia (aCML) in a Cohort of 43 Patients

Blood ◽

10.1182/blood-2019-126467 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1714-1714

Author(s):

Diletta Fontana ◽

Daniele Ramazzotti ◽

Andrea Aroldi ◽

Antonio Niro ◽

Luca Massimino ◽

...

Keyword(s):

Gene Expression ◽

Overall Survival ◽

Board Of Directors ◽

Rna Sequencing ◽

Myeloid Leukemia ◽

Sequencing Data ◽

Advisory Committees ◽

Atypical Chronic Myeloid Leukemia ◽

Worse Prognosis

Atypical chronic myeloid leukemia (aCML) is a rare BCR-ABL1 negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in several genes including SETBP1, ASXL1 and ETNK1, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. The clinical prognosis for aCML is poor, with a median overall survival of 18 months after diagnosis, and no established standards of care exist for its treatment. The dissection of the molecular processes underlying aCML leukemogenesis could therefore result decisive in ameliorating the prognosis for aCML. With the aim to provide a comprehensive genomic characterization of aCML and to link the detected alterations with the clinical course of the disease, we applied a high-throughput sequencing strategy to 43 aCML samples, including whole-exome sequencing and RNA sequencing. Our study confirms ASXL1 and SETBP1 as the most frequently mutated genes with a total of 43.2% and 30.2%, respectively; ETNK1 mutations are observed in 14% of patients. An average of 2 mutations per patient was observed [range: 0-5]. We characterized the clonal architecture in a subset of 8 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur very early in the clonal evolution history of aCML, while SETBP1 mutations represent a late event; interestingly, in the two cases where ASXL1 was mutated together with SETBP1, its mutations occupied an intermediate hierarchical position. CBL mutations, when present, showed a tendency toward reaching homozygosity through somatic uniparental disomy. Stratification based on RNA-sequencing gene expression data (Ramazzotti, Daniele, et al. Nature communications 9.1 (2018): 4453) identified two clearly different populations (26 and 17 patients) in terms of Overall Survival (OS), with 2 year OS of 69.23% [95% IC: 48.21%-86.67%] and 35.29% [95% IC: 14.21%-61.67%] respectively (logrank test for trend: p=0.004, Fig. 1A). In addition, the group with better prognosis showed a higher frequency of ETNK1 mutations (hypergeometric test: p=0.032). We next performed differential gene expression analysis to detect genes differentially expressed between the two patients' populations. This analysis revealed 38 significant genes (t-test p-value adjusted for false discovery rate p<0.01) overexpressed in the group with negative outcome. Notably, the majority of these genes are known cancer drivers, such as IDH2, MEN1, MYC and TP53. Involved pathways include gene transcription and cell differentiation, mitochondrial activity and DNA repair. We then considered RNA-sequencing data for the 4 most significant genes within the previous list (namely DNPH1, GFI1B, PARP1 and POLRMT) to build a classifier capable of associating patients to the respective subtype (better vs. worse prognosis). Our results show that a random forest classifier (Ho, Tin Kam. Proceedings of 3rd international conference on document analysis and recognition. Vol. 1. IEEE, 1995) using the 4 most significant genes achieves a 93.79% accuracy assessed by means of 10 fold cross validation (Fig.1B-C). In conclusion, we present here the first description of a large aCML cohort, in which sequencing data, clonal hierarchy of mutations and gene expression profiles were integrated through bioinformatics analysis. RNA-sequencing data stratification characterizes two groups with different prognosis; a classifier based on the 4 top differently expressed genes accurately predicts patients' outcome. Figure 1. A) Overall Survival curve (Kaplan-Meier curve) at 24 months shows significant different outcomes (p=0.004). B) Random forest classifiers learn multiple decision trees in order to predict outcomes. In the figure, an example of decision tree where nodes are genes and leaves are outcomes (better/worse prognosis). C) Heatmap of expression fold change for the top four differentially expressed genes. Figure 1 Disclosures Rea: BMS: Honoraria; Incyte Biosciences: Honoraria; Novartis: 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. Stagno:Pfizer: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Elli:Novartis: Membership on an entity's Board of Directors or advisory committees. Gambacorti-Passerini:Pfizer: Honoraria, Research Funding; Bristol-Meyers Squibb: Consultancy.

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The Genomic and Epigenomic Landscapes of Blast Crisis Transformation in Chronic Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.3737.3737 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3737-3737 ◽

Cited By ~ 3

Author(s):

S. Tiong Ong ◽

Thushangi Pathiraja ◽

Asif Javed ◽

Xin Xuan Sheila Soh ◽

Simeen Malik ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Chronic Myeloid Leukemia ◽

Board Of Directors ◽

Candidate Genes ◽

Myeloid Leukemia ◽

Research Funding ◽

Blast Crisis ◽

Advisory Committees ◽

Cpg Sites

Abstract The transition from chronic phase (CP) to blast crisis (BC) chronic myeloid leukemia (CML) is characterized by reprogramming of the CML transcriptome (Radich et al. PNAS 2006), and shortened survival. Current models propose genomic instability as causal in BC transformation with enhanced DNA damage and impaired DNA repair inducing genetic mutations (ranging from large chromosomal aberrations to point mutations), altered gene function, and eventually BC transformation (Perrotti et al. JCI 2010). Consistent with this model are the phenomena of BC clonal evolution, and the increased frequency of ABL kinase domain mutations found in BC. Because different mutational processes are associated with distinct cancer-specific mutation signatures (Alexandrov et al. Nature 2013), this model also predicts the existence of a CML-specific mutation signature. In addition, recent work has highlighted the importance of epigenetic alterations in hematologic malignancies (Shih et al., Nat. Rev. Cancer, 2012). However, we lack a complete understanding of the type or frequency of genetic alterations in BC, and the relative contribution of genetic vs. epigenetic events in reprogramming the BC transcriptome. To address these knowledge gaps, we analyzed the CML progression genome, epigenome, and transcriptome in 12 CP/BC sample pairs. Whole-genome sequencing revealed the CML genome to be relatively stable with respect to structural variations, indels, and somatic single nucleotide variants. The average number of nonsynonymous coding mutations per BC genome was 5, placing the BC coding genome in the same mutation frequency range as AML and ALL genomes (Alexandrov et al. Nature 2013). In addition, we identified a novel mutation signature in all CML samples suggesting a CML-specific mutational process. 1175 genes were 'hit' by genomic, mostly copy number, alterations in >1 sample, and included TCR genes and Ikaros (IKZF1) among lymphoid BC pairs. Only 21 recurrently altered genes were affected by somatic SNVs or indels, with resistance-associated ABL1 mutations being commonest. We next used DNA methylation arrays to assess the BC epigenome, and found 20,651 CpG sites (out of 455,187) to be hyper-methylated, and 3225 to be hypo-methylated in BC compared to CP. Combined methylome and transcriptome analysis demonstrated an inverse relationship between methylation and expression changes at a subset of CpG sites enriched at promoters. Genes with increased methylation/decreased expression or decreased methylation/increased expression included those involved in cell cycle control/heme biosynthesis, and molecular mechanisms of cancer/G-protein coupled receptor signaling/MAPK signaling respectively. Unsupervised methylation-based clustering segregated samples into CP, lymphoid BC and myeloid BC groups, recapitulating expression-based clustering, and further supporting a functional role for DNA methylation in BC transcriptional reprogramming. We next performed an integrative analysis by combining the genome, methylome, and transcriptome datasets, and included data from 34 additional CML samples. Top ranking candidate genes included epigenetic modifiers, and hematopoetic differentiation- and stem cell-related genes. Functional analysis of candidate genes and epigenetic processes using genetic and epigenetic drug-based approaches are ongoing. In summary, we conclude that: 1. The genomic and epigenomic landscapes in BC are characterized by a modest number of recurring events in the former, but consistent and striking differences in the latter, 2. The BC methylome is functionally associated with the robust gene expression changes found in BC, and 3. Epigenetic modifier drugs may be of use in reversing the gene expression changes characteristic of BC. Disclosures Chuah: Children International: Honoraria; Novartis: Honoraria; Bristol Meyers Squibb: Honoraria. Takahashi:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sysmex: Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Masis: Consultancy; Otsuka: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau.

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Alternative Overexpression of NRF2 or MYC Defines a Subgroup of Poor Prognosis Acute Myeloid Leukemia and Suggests a Novel Therapeutic Strategy By Combined Bromodomain Inhibition and Forced NRF2 Pathway Activation

Blood ◽

10.1182/blood-2018-99-117429 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2639-2639

Author(s):

Giorgia Simonetti ◽

Samantha Bruno ◽

Carmine Onofrillo ◽

Cristina Papayannidis ◽

Giovanni Marconi ◽

...

Keyword(s):

Gene Expression ◽

Cell Viability ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Poor Prognosis ◽

Advisory Committees ◽

Nrf2 Pathway ◽

Nrf2 Activation ◽

Acute Myeloid

Abstract Introduction. Inhibition of Bromodomain and extraterminal (BET) proteins was effective against different acute myeloid leukemia (AML) subtypes in preclinical studies (Dawson et al. Nature 2011; Zuber et al. Nature 2011; Dawson et al. Leukemia 2013; Chen et al. Cancer Cell 2014; Gröschel et al. Cell 2014; Zhao et al. Cell Reports 2016). However, the drug had limited clinical activity, suggesting the need of ad hoc combination therapies able to target leukemia stem cells (LSCs) in their microenvironment. Hypoxia is an integral component of the bone marrow microenvironment and plays a crucial role in survival and chemoresistance of LSCs. Aims. The study aims to elucidate the consequences of BETi treatment in AML under hypoxic conditions and identify novel potential combination strategies. Methods. AML cell lines (OCI-AML3: NPM1- and DNMT3A-mutated, Kasumi-1: t(8;21), HL60: MYC-amplified, MOLM-13, NOMO-1: MLL-driven, KG-1: TP53-mutated) were treated with the BET inhibitor (i) GSK1215101A (250/500 nM, 48h) or the NRF2 activator omaveloxolone (NRF2a, 0.2-1 mM, 48h) and with the drug combination (72h) at 1% or 20% O2 concentration. Cell viability, apoptosis and cell cycle were evaluated by trypan blue dye exclusion assay, AnnexinV and PI staining, respectively. Gene expression profiling (HTA2.0, Affymetrix) was carried out on actively translated mRNAs isolated by polysome profiling after 16h of BETi treatment and on 61 primary AML. The TCGA AML dataset was analyzed on the cBioPortal. Gene expression correlation and enrichment analysis were performed by Pearson coefficient and GSEA, respectively. Kaplan-Meier survival curves were compared by Logrank test. Glutathione was quantified by mass spectrometry (Metabolon). Results. BETi induced a dose-dependent reduction of cell viability in AML cells lines under hypoxia (25%-65% decrease at 500 nM) except for HL-60. Under the same conditions, the treatment caused a significant arrest in the G0/G1 phase of the cell cycle in OCI-AML3, Kasumi-1, HL-60 and KG-1 models (p<0.05) and induction of apoptosis in NOMO-1 and Kasumi-1 (40% and 50% AnnexinV+ cells, respectively, p<0.05). BETi reduced the translational rate of Kasumi-1 and OCI-AML3 cells, as determined by a decrease of disome-polysome peaks height. The treatment exacerbated hypoxia-mediated MYC suppression and associated with downregulation of a MYC signature at translational level. Moreover, it induced upregulation of the NRF2 regulator ARNT (p=0.02) and the NRF2 targets CAT, EPHX1, FTH1, GSTM1, MGST1, PRDX1 (p<0.05) under normoxia and/or hypoxia, with reduced KEAP1 mRNA and protein specifically at 1% O2 (p=0.01). These results suggest stabilization of NRF2 protein and activation of the pathway, as strengthened by increased levels of reduced and oxidized glutathione in OCI-AML3 cells (p<0.01). Based on this alternative activation of MYC and NRF2 pathway in AML, we analyzed gene expression and mutation in non-M3 AML from an internal cohort and the TCGA dataset. Upregulation of NRF2 expression and deregulation of MYC (overexpression/driver mutation) occurred in 4% and 9% of cases, respectively (independent of genomic amplification), with mutual exclusivity and an inverse correlation (p=0.03). MYC or NRF2 alterations defined a subgroup of patients with poor overall survival (10 vs. 18.1 months, p=0.04) and progression-free survival (7.2 vs. 17 months, p=0.0006). We then asked whether antioxidant gene expression was a defense response under BETi pressure. However, pharmacological inhibition of NRF2 or glutathione biosynthesis failed to potentiate the anti-leukemic effects of BETi. Conversely, activation of NRF2 pathway, which is effective as single agent on AML cells, potentiates the effects of BETi treatment in non-M3 AML, with reduced cell viability and increased apoptosis. Conclusions. BET protein activity drives alternative NRF2 or MYC overexpression in AML, which defines a subgroup of patients with poor prognosis. NRF2 activation is finely tuned in AML, as both inhibition and activation of the pathway induce cell death. However, NRF2 activation specifically potentiates BETi treatment under hypoxia and normoxia, suggesting a novel combination therapy against AML LSCs. Supported by: EHA Non-Clinical Junior Research Fellowship, ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, Fondazione del Monte BO e RA project. Figure. Figure. Disclosures Cavo: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Martinelli:Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Abbvie: Consultancy; Roche: Consultancy; Ariad/Incyte: Consultancy; Jazz Pharmaceuticals: Consultancy; Janssen: Consultancy; Novartis: Speakers Bureau.

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Distinct Gene Expression Signatures in Patients with Richter's Syndrome and Chronic Lymphocytic Leukemia with Prior Exposure to Ibrutinib

Blood ◽

10.1182/blood-2020-136172 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 30-31

Author(s):

Hanyin Wang ◽

Shulan Tian ◽

Qing Zhao ◽

Wendy Blumenschein ◽

Jennifer H. Yearley ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Activation ◽

Expression Profiles ◽

Lymphocytic Leukemia ◽

B Cell Activation ◽

Advisory Committees ◽

Upregulated Genes

Introduction: Richter's syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into a highly aggressive lymphoma with dismal prognosis. Transcriptomic alterations have been described in CLL but most studies focused on peripheral blood samples with minimal data on RS-involved tissue. Moreover, transcriptomic features of RS have not been well defined in the era of CLL novel therapies. In this study we investigated transcriptomic profiles of CLL/RS-involved nodal tissue using samples from a clinical trial cohort of refractory CLL and RS patients treated with Pembrolizumab (NCT02332980). Methods: Nodal samples from 9 RS and 4 CLL patients in MC1485 trial cohort were reviewed and classified as previously published (Ding et al, Blood 2017). All samples were collected prior to Pembrolizumab treatment. Targeted gene expression profiling of 789 immune-related genes were performed on FFPE nodal samples using Nanostring nCounter® Analysis System (NanoString Technologies, Seattle, WA). Differential expression analysis was performed using NanoStringDiff. Genes with 2 fold-change in expression with a false-discovery rate less than 5% were considered differentially expressed. Results: The details for the therapy history of this cohort were illustrated in Figure 1a. All patients exposed to prior ibrutinib before the tissue biopsy had developed clinical progression while receiving ibrutinib. Unsupervised hierarchical clustering using the 300 most variable genes in expression revealed two clusters: C1 and C2 (Figure 1b). C1 included 4 RS and 3 CLL treated with prior chemotherapy without prior ibrutinib, and 1 RS treated with prior ibrutinib. C2 included 1 CLL and 3 RS received prior ibrutinib, and 1 RS treated with chemotherapy. The segregation of gene expression profiles in samples was largely driven by recent exposure to ibrutinib. In C1 cluster (majority had no prior ibrutinb), RS and CLL samples were clearly separated into two subgroups (Figure 1b). In C2 cluster, CLL 8 treated with ibrutinib showed more similarity in gene expression to RS, than to other CLL samples treated with chemotherapy. In comparison of C2 to C1, we identified 71 differentially expressed genes, of which 34 genes were downregulated and 37 were upregulated in C2. Among the upregulated genes in C2 (majority had prior ibrutinib) are known immune modulating genes including LILRA6, FCGR3A, IL-10, CD163, CD14, IL-2RB (figure 1c). Downregulated genes in C2 are involved in B cell activation including CD40LG, CD22, CD79A, MS4A1 (CD20), and LTB, reflecting the expected biological effect of ibrutinib in reducing B cell activation. Among the 9 RS samples, we compared gene profiles between the two groups of RS with or without prior ibrutinib therapy. 38 downregulated genes and 10 upregulated genes were found in the 4 RS treated with ibrutinib in comparison with 5 RS treated with chemotherapy. The top upregulated genes in the ibrutinib-exposed group included PTHLH, S100A8, IGSF3, TERT, and PRKCB, while the downregulated genes in these samples included MS4A1, LTB and CD38 (figure 1d). In order to delineate the differences of RS vs CLL, we compared gene expression profiles between 5 RS samples and 3 CLL samples that were treated with only chemotherapy. RS samples showed significant upregulation of 129 genes and downregulation of 7 genes. Among the most significantly upregulated genes are multiple genes involved in monocyte and myeloid lineage regulation including TNFSF13, S100A9, FCN1, LGALS2, CD14, FCGR2A, SERPINA1, and LILRB3. Conclusion: Our study indicates that ibrutinib-resistant, RS-involved tissues are characterized by downregulation of genes in B cell activation, but with PRKCB and TERT upregulation. Furthermore, RS-involved nodal tissues display the increased expression of genes involved in myeloid/monocytic regulation in comparison with CLL-involved nodal tissues. These findings implicate that differential therapies for RS and CLL patients need to be adopted based on their prior therapy and gene expression signatures. Studies using large sample size will be needed to verify this hypothesis. Figure Disclosures Zhao: Merck: Current Employment. Blumenschein:Merck: Current Employment. Yearley:Merck: Current Employment. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Parikh:Verastem Oncology: Honoraria; GlaxoSmithKline: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Kenderian:Sunesis: Research Funding; MorphoSys: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Gilead: Research Funding; BMS: Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Juno: Research Funding; Mettaforge: Patents & Royalties; Torque: Consultancy; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding. Kay:Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ding:DTRM: Research Funding; Astra Zeneca: Research Funding; Abbvie: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees.

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Identifying Factors That Predict for Unplanned Readmissions for Acute Myeloid Leukemia Patients Receiving Consolidation Cytarabine Based Therapies

Blood ◽

10.1182/blood-2019-128807 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3433-3433

Author(s):

Caitlin Siebenaller ◽

Madeline Waldron ◽

Kelly Gaffney ◽

Brian P. Hobbs ◽

Ran Zhao ◽

...

Keyword(s):

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Smoking Status ◽

Consolidation Therapy ◽

Peripheral Vascular ◽

Consolidation Chemotherapy ◽

Advisory Committees ◽

History Of ◽

Acute Myeloid

Background: Younger patients (pts) with acute myeloid leukemia (AML) who enter a remission after intensive induction chemotherapy routinely receive at least one cycle of consolidation therapy with high dose cytarabine (HiDAC). This is commonly administered over a five-day inpatient stay, after which pts are discharged home as their blood counts nadir. It is thus a natural consequence of therapy that readmission for febrile neutropenia (FN) occurs, which can impact measures of quality and value in this population. Precise descriptions of incidence, type, and severity of infection, if identified, are lacking, and thus it is unknown to what standard cancer centers should be held for anticipated readmission. We measured these rates, and attempted to identify predictive factors for readmission. Methods: Adult AML pts ≥ 18 years of age who received at least one cycle of HiDAC consolidation (1000-3000 mg/m2 for six doses) in 2009-2019 were included. Our primary aim was to identify predictive factors for readmission after the first cycle of consolidation chemotherapy. The following pt characteristics and co-morbid conditions were analyzed: age, gender, body mass index (BMI), smoking status, AML cytogenetic risk status, history of diabetes, peripheral vascular disease, cardiovascular disease, chronic pulmonary disease, hepatic impairment, and other cancers. Secondary aims included: estimating rates of all-cause readmissions among all HiDAC cycles, defining the rate of FN readmissions, estimating rates of intensive care unit (ICU) admissions, clinical (e.g., probable pneumonia per imaging) and microbiologically-documented infections, prophylactic (ppx) medications used, and mortality. Statistical analyses interrogated potential risk factors for evidence of association with hospital readmission after the first cycle of consolidation chemotherapy. Results: We identified 182 AML pts who fit inclusion criteria. The median age was 50 years (range 19-73); 55% were female and 45% were male. Statistical analyses revealed no association with readmission after cycle 1 for cytogenetic risk (p=0.85), history of heart failure (p= 0.67), chronic pulmonary disease (p=1), connective tissue disease (p=0.53), cerebrovascular accident (p=0.63), diabetes (p=0.63), gender (p=0.07), history of lymphoma (p=0.53), other solid tumors (p=0.53), liver disease (p=1), myocardial infarction (p=0.71), peripheral vascular disease (p=1), or smoking status (p= 0.52). For 480 HiDAC cycles analyzed (88% at 3000 mg/m2), the overall readmission rate was 50% (242/480), of which 85% (205/242) were for FN. Those readmissions which were not FN were for cardiac complications (chest pain, EKG changes), non-neutropenic fevers or infections, neurotoxicity, bleeding or clotting events, or other symptoms associated with chemotherapy (nausea/vomiting, pain, etc.). Median time to FN hospital admission was 18 days (range 6-27) from the start of HiDAC. Of the 205 FN readmissions, 57% had documented infections. Of these infections, 41% were bacteremia, 23% fungal, 16% sepsis, 12% other bacterial, and 8% viral. Of 480 HiDAC cycles, ppx medications prescribed included: 92% fluoroquinolone (442/480), 81% anti-viral (389/480), 30 % anti-fungal (142/480), and 3% colony stimulating factor (14/480). Only 7% (14/205) of FN readmissions resulted in an ICU admission, and 1% (3/205) resulted in death. Conclusions: Approximately half of patients treated with consolidation therapy following intensive induction therapy can be expected to be readmitted to the hospital. The majority of FN readmissions were associated with clinical or microbiologically documented infections and are not avoidable, however ICU admission and death associated with these complications are rare. Readmission of AML pts following HiDAC is expected, and therefore, should be excluded from measures of value and quality. Disclosures Waldron: Amgen: Consultancy. Hobbs:Amgen: Research Funding; SimulStat Inc.: Consultancy. Advani:Macrogenics: Research Funding; Abbvie: Research Funding; Kite Pharmaceuticals: Consultancy; Pfizer: Honoraria, Research Funding; Amgen: Research Funding; Glycomimetics: Consultancy, Research Funding. Nazha:Incyte: Speakers Bureau; Abbvie: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee; Tolero, Karyopharma: Honoraria. Gerds:Imago Biosciences: Research Funding; Roche: Research Funding; Celgene Corporation: Consultancy, Research Funding; Pfizer: Consultancy; CTI Biopharma: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Sierra Oncology: Research Funding. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Mukherjee:Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy; McGraw Hill Hematology Oncology Board Review: Other: Editor; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees.

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Patient Characteristics and Outcomes in Adolescents and Young Adults (AYA) with Acute Myeloid Leukemia (AML): Princess Margaret Cancer Centre Experience

Blood ◽

10.1182/blood-2019-125449 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2609-2609

Author(s):

Muhned Alhumaid ◽

Georgina S Daher-Reyes ◽

Wilson Lam ◽

Arjun Law ◽

Tracy Murphy ◽

...

Keyword(s):

Board Of Directors ◽

Older Patients ◽

Myeloid Leukemia ◽

Research Funding ◽

Age Groups ◽

Treatment Strategies ◽

Patient Characteristics ◽

Cancer Center ◽

Advisory Committees ◽

Disease Behavior

Introduction: Clinical outcomes of acute myeloid leukemia (AML) in adolescents and young adults (AYA) are rarely reported as an isolated subgroup. Treatments vary little across age groups, and treatment intensity depends upon comorbid conditions and performance status. Optimal treatment strategies focused on disease behavior, biological factors, and the distinct needs of this subset of AML patients remain elusive. The purpose of this retrospective analysis is to determine the characteristics and outcomes of AYA AML patients treated at a specialized adult leukemia cancer center in comparison to older adults with AML (40-60 years). Methods: A retrospective analysis was performed on all patients treated at Princess Margaret Cancer Center from 2008-2018. Patients with acute promyelocytic leukemia were excluded. Clinical characteristics, treatment strategies, and survival outcomes were recorded for all patients. Overall survival (OS) and disease-free survival (DFS) rates were calculated using the Kaplan-Meier product-limit method and the impact of covariates were assessed using the Log-rank test. Finally, we compared the outcomes of AYA patients treated at our centre between 2015-2018 with older patients. Results: A total of 175 patients aged 18-39 were identified. Patient characteristics are shown in (Table 1). Cytogenetic were available in 163 patients. Based on MRC criteria, 27 (16%) were favorable risk, intermediate in 95 (54%), adverse in 39 (22%), and missing/failed in 14(8%). NPM1 status was available in 110 patients of whom 38 (35%) were positive. FLT3-ITD was available in 67 patients with 24 (36%) positive. Both mutations were present in 13 (54%) patients. There were no significant differences in terms of risk stratification based on cytogenetic and molecular markers based on age (18-29 vs.30-39) (P= 0.98). Most patients 172 (98%) received induction, 157 (91%) with 3+7, and 15 (9%) with FLAG-IDA. Complete remission (CR) was achieved in 133 (77%) after first induction [120 (76%) after 3+7 and 11 (73%) after FLAG-IDA]. Induction related mortality was low (2%). Of the 39 who did not achieve CR, thirty-four patients received re-induction (13 FLAG-IDA, 16 NOVE-HiDAC, 5 others) with CR in 21 (62%). Overall, 154 (89.5%) achieved CR1. Sixty-four (42%) proceeded to hematopoietic stem cell transplantation (HSCT) in CR1. 59 (38%) patients relapsed in CR1 with 8 (12%) relapsing post HSCT. Fifty-five (5 post HSCT) patients received reinduction with 30 (51%) (2 after HSCT) achieving CR2. Fifteen patients received HSCT in CR2. OS and DFS at 2 years were 62% (95% CI 0.53-0.69) and 50% (95% CI 0.41-0.57), respectively. Stratified by cytogenetic risk, OS was 81% for favorable risk, 61% for intermediate, and 50% for adverse risk (P=0.0001), respectively. DFS in these groups was 85%, 57%, and 46 % (P=0.0025), respectively. We further compared outcomes in the 18-29y and 30-39y age groups. The OS was 61.9% compared to 62.5% (P=0.91) and DFS of 52.1% compared to 47% (P=0.65) respectively. On univariate analysis for OS and DFS, cytogenetic risk stratification was the only significant variable (P=0.0004 and P=0.0042). We then compared the outcomes 67 sequential patients aged I8-39 treated from 2014-2018, with those of 176 sequential patients aged 40-60 treated during the same period (table 2). OS at 2 years was not statistically higher in the younger group compared to the older group (66.7% vs. 61.2%, P=0.372). While relapse rate was lower in older patients (15.5% vs. 22.6%, P=0.093), NRM was higher in older patients (29.7% vs. 18.8%,P=0.094). Conclusion: AYA pts. occupy a unique niche amongst AML as a whole. While treatment responses have improved in general, there may be potential for further gains in these patients. Increased tolerance for more intense treatment strategies as well as the incorporation of novel agents into standard treatment protocols may provide a means to optimize care in AYA patients. Finally, research is needed to elucidate biological mechanisms and predictors of disease behavior instead of arbitrary, age-stratified treatment schema. Disclosures McNamara: Novartis Pharmaceutical Canada Inc.: Consultancy. Schimmer:Jazz Pharmaceuticals: Consultancy; Medivir Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy. Schuh:Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: 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; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Teva Canada Innovation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees. Maze:Pfizer Inc: Consultancy; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yee:Astellas: Membership on an entity's Board of Directors or advisory committees; Millennium: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Astex: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MedImmune: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Hoffman La Roche: Research Funding. Minden:Trillium Therapetuics: Other: licensing agreement. Gupta:Incyte: Honoraria, Research Funding; Celgene: 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; Sierra Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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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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