Subset Of Acute Myeloid Leukemia Patients Research Articles

Chromoanagenesis Is Frequently Associated With Highly Complex Karyotypes, Extensive Clonal Heterogeneity, and Treatment Refractoriness in Acute Myeloid Leukemia.

Chromoanagenesis (CAG) encompasses a spectrum of catastrophic genomic events, including chromothripsis, chromoanasynthesis, and chromoplexy. We studied CAG in 410 patients with a diagnosis of acute myeloid leukemia (AML), 292 newly diagnosed (ND), and 118 refractory/relapsed, using optical genome mapping. CAG was identified by the presence of clusters (with 10 or more breakpoints) of structural abnormalities and/or segmental copy number alterations within one or more chromosomal regions. CAG was detected in 65 (16%) patients. Compared with patients without CAG, those with CAG showed significantly (p < 0.0001) higher frequencies of highly complex karyotype (92% vs. 11%), monosomal karyotype (88% vs. 12%), extensive clonal heterogeneity (75% vs. 7%), gene amplification (49% vs. 1%), and TP53 deletion/mutation (92% vs. 9%). Overall, CAG was detected in about two-thirds of AML patients who exhibited the abovementioned high-risk cytogenetic abnormalities/karyotype. Among the 42 patients with ND AML and CAG, 36 received treatments and follow-ups, and 28 (78%) had no or only partial response to therapy. Among patients with ND AML, those with CAG had a shorter overall survival than those without CAG (median survival: 5 vs. 14 months, p < 0.0001). However, in multivariate analysis, CAG did not appear to be an independent risk factor for survival. These results indicate that CAG is frequently associated with high-risk chromosomal alterations and genomic instability in AML and may contribute to treatment refractoriness and inferior survival in this subset of AML patients.

Abstract IA023: Targeting epigenetic complexes in leukemia

Abstract Chromatin-associated multi-protein complexes, which often comprise proteins involved in epigenetic regulation, play an important role in various cancers, including leukemia. Thus, pharmacologic inhibition of proteins involved in these complexes can lead to new anti-cancer therapies. We have focused on therapeutic targeting of various chromatin-bound multi-protein complexes, including Mixed Lineage Leukemia 1 (MLL1) and Polycomb Repressive Complex 1 (PRC1) complexes, as a novel treatment for leukemia. These efforts led to the development of small molecule inhibitors targeting the menin-MLL1 interaction, which were translated to leukemia patients, demonstrating promising efficacy in Acute Myeloid Leukemia (AML) with NPM1 mutations or MLL1 translocations. However, clinical studies revealed resistance to the single agent menin inhibitor in a sub-set of AML patients, suggesting that combinatorial treatments or new generations of menin inhibitors might be required to overcome resistance. Our recent efforts to address resistance to menin inhibitors will be presented, including combinatorial studies with other targeted agents (e.g. kinases inhibitors), which resulted in synergistic effects. Furthermore, discovery of a new generation of menin inhibitors with promising activity against menin patient mutations will also be presented. Citation Format: Jolanta Grembecka. Targeting epigenetic complexes in leukemia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA023.

Abstract 3229: IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets

Abstract Block in differentiation and accumulation of undifferentiated blasts are hallmarks of Acute Myeloid Leukemia (AML) and epigenetic processes have been shown to play a critical role in hematological malignancies. Paralogs CREBBP and EP300, in association with co-factors, are hijacked during leukemogenesis by aberrant transcriptional factors, thus driving blast proliferation and maintaining the undifferentiated phenotype. Here, we introduce IACS-16559, a bromodomain inhibitor targeting CREBBP/EP300. IACS-16559 is highly selective for the bromodomains of CBP and EP300 versus BRD4, showing a dose-dependent inhibition of H3K27 acetylation residue in vivo and in vitro. In addition, IACS-16559 pharmacokinetic properties show low clearances, high oral exposures and predicted QD dosing in human. We screened AML cell lines in vitro for their response to IACS-16559 and identified specific subtypes of AML that exhibit heightened sensitivity to CBP/EP300 inhibition, specifically AML with AML1-ETO and MLL-AF9 rearrangements, and AML featuring mutations in the nucleophosmin (NPM1c) gene. IACS-16559 inhibited proliferation of AML responder cell lines in vitro, halted cell cycle progression, with an accumulation of cells in G1 and a decrease of cells in S phase. IACS-16559 did not induce apoptosis across a panel of AML cell lines, indicating that induction of apoptosis is not a major mechanism of action of CBP/EP300 inhibition. To augment the anti-tumor effects of IACS-16559, we prioritized the pharmacological combination of CBP/EP300 and MLL-Menin inhibitors. This combination synergistically impeded cell proliferation and potently triggered apoptosis in vitro and in vivo, whereas none of the individual agents alone had this effect. The changes in gene and surface marker expression produced by these treatments, as single agents and in combination with synergistic effects, were in line with an induced myeloid differentiation phenotype. Collectively, our findings provide a compelling basis for further exploring the use of a CBP/EP300 bromodomain inhibitor in conjunction with an inhibitor of the MLL-Menin interaction in a subset of AML patients with MLL rearrangements or NPM1 mutations. Citation Format: Ciro Zanca, Michael Soth, Guang Gao, Jason Gay, Ningping Feng, Christopher Bristow, Kang Le, Quanyun Xu, Phuong Nguyen, Yongying Jiang, Teresa Perry, Faika Mseeh, Paul Leonard, Jason Cross, Virginia Giuliani, Joe Marszalek, Philip Jones, Tim Heffernan. IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3229.

Inhibition of Enhancer of Zeste Homolog 2 Induces Blast Differentiation, Impairs Engraftment and Prolongs Survival in Murine Models of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the malignant proliferation of immature myeloid cells characterized by a block in differentiation. As such, novel therapeutic strategies to promote the differentiation of immature myeloid cells have been successful in AML, although these agents are targeted to a specific mutation that is only present in a subset of AML patients. In the current study, we show that targeting the epigenetic modifier enhancer of zeste homolog 2 (EZH2) can induce the differentiation of immature blast cells into a more mature myeloid phenotype and promote survival in AML murine models. The EZH2 inhibitor EPZ011989 (EPZ) was studied in AML cell lines, primary in AML cells and normal CD34+ stem cells. A pharmacodynamic assessment of H3K27me3; studies of differentiation, cell growth, and colony formation; and in vivo therapeutic studies including the influence on primary AML cell engraftment were also conducted. EPZ inhibited H3K27me3 in AML cell lines and primary AML samples in vitro. EZH2 inhibition reduced colony formation in multiple AML cell lines and primary AML samples, while exhibiting no effect on colony formation in normal CD34+ stem cells. In AML cells, EPZ promoted phenotypic evidence of differentiation. Finally, the pretreatment of primary AML cells with EPZ significantly delayed engraftment and prolonged the overall survival when engrafted into immunodeficient mice. Despite evidence that EZH2 silencing in MDS/MPN can promote AML pathogenesis, our data demonstrate that the therapeutic inhibition of EZH2 in established AML has the potential to improve survival.

Fever with Significant Procalcitonin Increase Following Gemtuzumab Ozogamicin Infusion in Hematologic Malignancy Patients without Evidence of Infection

In patients diagnosed with hematologic malignancies, including acute myeloid leukemia (AML), infections remain a major cause of non-relapse mortality. The accurate detection and management of infections during the treatment of these diseases are crucial for successful outcomes. Procalcitonin (PCT) has been proposed as a valuable tool for identifying severe bacterial infections, even in patients with hematologic malignancies. Gemtuzumab ozogamicin (GO), a humanized anti-CD33 monoclonal antibody conjugated to calicheamicin, is used in certain subsets of AML patients, such as core binding factor (CBF) leukemia and AML with favorable-risk, as well as in cases of refractory AML. However, GO administration comes with its own set of risks, including liver dysfunction, veno-occlusive disease (VOD), bone marrow suppression, bleeding, and infusion reactions. Among these reactions, those causing high fever and shivering can be particularly challenging to differentiate from sepsis or severe infections. Nevertheless, there have been no reports evaluating the utility of PCT in discriminating infections during GO-induced infusion reactions. To shed light on the impact of GO administration on PCT levels, we undertook a retrospective analysis. Over the period between 2014 and 2022, we administered GO (3.0 mg/sqm per dose) to 42 patients with hematologic malignancies, a total of 97 times (median 2 doses per patient, range 1-6 doses per patient). Of the recipients, 23 had undergone allogeneic stem cell transplantation, while 19 were non-transplant patients. Prior to GO administration, all patients received premedication comprising corticosteroids, antihistamines, and acetaminophen. Among the 97 instances of GO administration, 40 (41.2%) resulted in fever (≥37.5 ℃) within 24 hours. For 19 cases, we conducted a complete set of PCT measurements and blood cultures within 24 hours of GO administration and analyzed the PCT levels and clinical parameters. These 19 cases corresponded to 19 individual patients, of whom nine had CBF leukemia, including t(8;21) AML or inv(16) AML, and ten had non-CBF leukemia. Out of these patients, 12 experienced fever (≥37.5 ℃), while seven did not. The findings indicated that patients with fever (≥37.5 ℃) exhibited significantly higher PCT levels compared to afebrile patients (p=0.018). Furthermore, PCT levels showed a significant positive correlation with C-reactive protein levels (p=0.003). However, among the 19 patients, only one patient with a positive PCT result had a documented infection identified through blood cultures or imaging studies.Other factors, such as patient gender (p=0.467), underlying disease (p=0.224), disease remission status at GO administration (p=0.492), transplant status (p=0.556), and the number of GO administrations (p=0.906), did not significantly influence PCT levels. It is essential to acknowledge that this analysis is based on a limited number of cases from a single institution. Nevertheless, approximately 40% of patients experienced fever within 24 hours of GO administration, despite receiving premedication.Furthermore, the presence of fever after GO administration was associated with a significant increase in PCT levels, even in the absence of identifiable infection. This raises the possibility of false positives for PCT in the context of GO-induced infusion reactions, underscoring the need for cautious interpretation of PCT values for infection differentiation in patients undergoing regimens that include GO. Further research with larger patient cohorts is warranted to validate these findings and improve our understanding of the implications of PCT measurements in this clinical setting.

Abstract 1594: SMARCA2 (BRM) degraders promotes differentiation and inhibit proliferation in AML models

Abstract Dysregulated cellular differentiation is a major pathological feature of myeloid malignancies such as acute myeloid leukemia (AML). Targeting cellular differentiation programs has emerged as a novel therapeutic approach to treat patients with AML. Advantages of such differentiation therapy may include fewer systemic side-effects as well as opportunities to target leukemic stem cells (LSCs) and a broader range of clonal populations, likely resulting in lower frequencies of resistance and relapse in AML patients. The success of ATRA and decitabine in subsets of AML patients has proven that inducing differentiation can play a critical role in long-term durable responses. More agents targeting epigenetic regulators have been increasingly studied as differentiation inducers, including LSD1, DNMT1, Menin, and BET inhibitors. Recently, targeting SWI/SNF chromatin remodeling complexes has also been shown to regulate key leukemic gene expression signatures and induce AML differentiation. Small molecule inhibitors as well as gene knockdown for ATP-dependent SWI/SNF subunits SMARCA2 (BRM) and SMARCA4 (BRG1) are associated with re-direction of oncogenic transcriptional regulation to drive cellular differentiation and apoptosis in AML models. We have previously described the activity of highly potent, bispecific SMARCA2 degraders that efficiently promote SMARCA2 protein degradation in preclinical models. In the present study, we investigated the effects of our SMARCA2 selective degraders in AML models. Treatment with SMARCA2 degraders significantly inhibits AML cell line proliferation in vitro with IC50 ranging from 10 to 50 nM. In the SMARCA2 degrader treated cells, expression of PU.1 (SPI1), a key transcription factor in myeloid leukemias, was downregulated. In an in vivo OCI-AML3 xenograft model, treatment with a SMARCA2 selective degrader showed moderate tumor growth inhibition accompanied by robust increases in monocytic maturation markers CD11b and CD14. Further analyses of SMARCA2 degrader effects on global transcriptome and AML immunophenotypes as well as combination effects with other therapies are currently in progress. These findings highlight the potential of SMARCA2 degraders to target AML differentiation blocks and to improve the effectiveness of other therapeutic agents such as decitabine and venetoclax in AML patients. Citation Format: Anjana Agarwal, Olusola Peace Osinubi, Komali Vykuntam, Norman Fultang, Neha Bhagwat, Diane Heiser, Kris Vaddi, Koichi Ito, Peggy Scherle. SMARCA2 (BRM) degraders promotes differentiation and inhibit proliferation in AML models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1594.

Smoking status in acute myeloid leukemia is associated with worse overall survival and independent of prior nonhematopoietic malignancies, cytogenetic abnormalities, and WHO category

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy with several patient- and disease-associated variables known to impact prognosis. Tobacco smoking is an environmental factor associated with a greater incidence of AML, but there have been limited studies that evaluated smoking toward overall survival. We retrospectively searched for AML cases and collected clinical and diagnostic data for each case. We also used an independent next-generation sequencing (NGS) data set to assess for a distinct mutational signature associated with smoking. When stratified by smoking status, there was a greater number of males, patients aged ≥60 years, and patients with ≥2 comorbidities within the smoking category (P<.05). Survival analysis demonstrated decreased survival probability in the smokers, male smokers, smokers with 1 other comorbidity, and smokers without a prior history of nonhematopoietic malignancy (P<.05) as compared to nonsmokers. Smoking was associated with a decrease in survival within the World Health Organization categories of AML, not otherwise specified (AML NOS; P=.035) and AML with recurrent genetic abnormalities (AML RGA; P=.002). Multivariate analysis showed that patients who were smokers had a greater hazard ratio than nonsmokers after adjusting for the other covariates. Our findings demonstrated that smoking was independently associated with decreased overall survival after adjusting for other potentially confounding factors. In addition, our results suggest that a mutational signature can be recognized using NGS data in a subset of AML patients who smoke.

An inflammatory state remodels the immune microenvironment and improves risk stratification in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a hematopoietic malignancy with poor prognosis and limited treatment options. Here we provide a comprehensive census of the bone marrow immune microenvironment in adult and pediatric patients with AML. We characterize unique inflammation signatures in a subset of AML patients, associated with inferior outcomes. We identify atypical B cells, a dysfunctional B-cell subtype enriched in patients with high-inflammation AML, as well as an increase in CD8+GZMK+ and regulatory T cells, accompanied by a reduction in T-cell clonal expansion. We derive an inflammation-associated gene score (iScore) that associates with poor survival outcomes in patients with AML. Addition of the iScore refines current risk stratifications for patients with AML and may enable identification of patients in need of more aggressive treatment. This work provides a framework for classifying patients with AML based on their immune microenvironment and a rationale for consideration of the inflammatory state in clinical settings.

The Impact of Chronic Kidney Disease on Hospitalization Outcomes in Patients Admitted with Acute Myeloid Leukemia: A Nationwide Analysis Using the National Inpatient Sample Database (2015-2019)

Background: Patients with baseline chronic kidney disease (CKD) are more susceptible to acute kidney injury during treatment for acute myeloid leukemia (AML). In addition, some CKD patients may not be a candidate for intensive therapeutic regimens due to their comorbidities. These factors increase the risk of complications during hospitalization for this subset of AML patients. Methods: The data for this study was obtained from the publicly available National Inpatient Sample Database (NIS). We used the International Classification of Diseases, 10th Revision (ICD-10) codes to collect data on hospitalizations for AML for patients >18 years of age. The data was further classified into hospitalizations with or without CKD (all stages included). The Mann-Whitney U and Chi-square tests were performed to compare continuous and categorical variables respectively for the unadjusted data. Propensity Score Matching (PSM) was done to account for multiple potentially confounding demographic variables and co-morbid conditions such as age, gender, sex, chronic obstructive pulmonary disease (COPD), pulmonary hypertension, diabetes, hypothyroidism, obesity, coronary artery disease (CAD), history of myocardial infarction, smoking, liver disease, peripheral vascular disease (PVD), alcohol abuse, and drug abuse. A total of 4330 AML cases with CKD were matched with 4330 cases of AML without CKD (control group). A multivariate logistic regression model was then performed taking into consideration the hospital characteristics, median household income of the patients, and the previously mentioned comorbidities to generate the adjusted odds ratio for outcomes of interest between the two groups. Results: The data for 53,068 hospitalizations for AML was collected between 2015-2019. Among these, 6997 cases had concurrent CKD. Patients with CKD were significantly older than their counterparts (72 vs 63 years, p<0.001), were more likely to be male (65.8% vs 53.4%, p<0.001), and black (13.9% vs 9.6%, p<0.001). They also had significantly more comorbid conditions like CAD, COPD, history of myocardial infarction, hypertension, diabetes, hypothyroidism, liver disease, and PVD. After PSM, multivariate logistic regression revealed CKD patients with AML had significantly higher odds of need for hemodialysis (OR: 4.26, 95% CI: 2.94-6.17, p<0.001), and mechanical ventilation (OR: 1.34, 95% CI: 1.13-1.58, p<0.001). AML patients with CKD also had higher odds of tumor lysis syndrome (OR: 2.00, 95% CI: 1.62-2.47; p<0.001), cardiogenic shock (OR: 3.55, 95% CI: 1.69-7.44, p<0.001), septic shock (OR: 1.38, 95% CI: 1.14-1.66, p<0.001), and all-cause mortality (OR: 1.29, 95% CI: 1.12-1.48, p<0.001). Interestingly, AML patients with CKD had lower odds of receiving a blood transfusion (OR: 0.87, 95% CI: 0.79-0.95; p=0.003), or platelet transfusion (OR: 0.79, 95% CI: 0.71-0.88; p<0.001) likely because of the lower chance of receiving intensive induction therapy as they had higher odds of receiving palliative care treatment instead (OR: 1.25, 95% CI: 1.12-1.48, p<0.001) (Figure 1). Conclusion: This large retrospective study suggests CKD as an independent risk factor for adverse hospitalization outcomes regardless of treatment modality in AML patients. CKD should therefore be accounted for when making therapy decisions for this subset of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Defining Clinical and Molecular Biomarkers for Venetoclax-Based Drug Combinations to Augment AML Therapy

Background: The combination of the BCL2 inhibitor venetoclax with azacytidine (Ven+Aza) is efficacious for elderly patients with acute myeloid leukemia (AML) who are unfit for standard chemotherapy. However, the overall and progression-free survival of these patients after 1 year of therapy is approximately 30-40%, and resistance to Ven+Aza, either primary or adaptive through clonal evolution, limits its durability. Therefore, we evaluated new venetoclax combinations to elucidate mechanisms and biomarkers of response and resistance. Methods: We tested a set of 31 venetoclax (Ven) combinations using an ex vivo assay on primary AML patient specimens (n=433, though not all combinations were screened for all samples). Enhanced efficacy was determined using a combination ratio (CR), defined as the sensitivity of the combination (by AUC) divided by the sensitivity of the most potent single agent AUC. Drug combination synergy was evaluated using the highest single agent (HSA) method. Clinical, genetic, and disease status information were collated, and ex vivo combination sensitivities were compared to matched single-agent data by Friedman test, across groups by Wilcoxon rank sum or Kruskal-Wallis test, and with continuous variables by Spearman rank correlation. For patient specimens with RNAseq data collected (n=279 patients), transcriptomic gene signatures of AML tumor cell state (van Galen, Cell 2019) were correlated with combination sensitivity by Spearman rank. Uni- and multi-variate analyses were applied to the full dataset to identify biomarkers of differential sensitivity to each combination. For one combination, Ven plus the JAK1/2 inhibitor ruxolitinib (Rux), additional genome wide CRISPR screens and follow up in vivo patient xenograft and mechanistic validation experiments using AML cell line models were performed. Results: Using Ven+Aza as a benchmark, we found several venetoclax combinations that exceeded both the ex vivo activity and enhanced efficacy observed with Ven+Aza in primary AML patient samples (Fig. 1A), while showing limited to no effect on stromal cell controls. Evaluation of clinical and genetic data with combination sensitivity revealed many significant transcriptomic, genetic, and immunophenotypic associations. The strong bias in sensitivity of Ven with respect to transcriptomic signatures of primitive tumor cell states compared to more mature, monocytic tumor states was unchanged with Ven+Aza and select other combinations. Importantly, this differential sensitivity was overcome when pairing Ven with several new drug partners such as Rux or the p38 MAPK inhibitor doramapimod (Fig. 1B). Uni- and multi-variate analyses further revealed an array of different biomarkers involving clinical and genetic features that correlated with significant differential sensitivity to each combination. Many of these likewise involved markers of cell differentiation state, such as CD14. To elucidate the mechanisms of enhanced efficacy of the Ven+Rux combination, which is currently under phase 1 clinical investigation, in vivo murine patient xenograft models and genome-wide CRISPR screens in OCI-AML2 cells were performed. Ven+Rux demonstrated superior inhibition of tumor growth in an AML xenograft model compared to either single-agent. The Ven+Rux CRISPR screen identified many genes whose knockdown was enriched in combination-resistant cells, including three (MDM2, MED13L, MEIS2) that were confirmed to confer resistance in subsequent immunoblotting and drug sensitivity validation experiments. In comparison, analysis of a control CRISPR screen using Ven alone revealed numerous gene hits in the JAK/STAT signaling pathway as enhancing the sensitivity of Ven. Conclusion: To expand the utility of venetoclax-inclusive combinations, we prospectively profiled a set of drug combinations and identified several with activity that exceeds Ven+Aza in ex vivo assays. Associations of clinical features with sensitivities to these drug combinations provide insight into their effectiveness which may be used to align the most promising combinations with translation to treatment of discrete subsets of AML patients. Together, these findings support opportunities for expanding the impact of Ven-based drug combinations in AML by leveraging clinical and molecular biomarkers of response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clofarabine-Based Consolidation Improves Relapse-Free Survival of Patients with Acute Myeloid Leukemia with Complex or Micro-Complex Karyotype: Results from the Randomized ALFA-0702 Study

Introduction. Acute myeloid leukemia (AML) encompasses heterogeneous entities, but global outcome remains poor. Among genetic AML subgroups, complex karyotypes represent one of the most difficult-to-treat. Genomic alterations arising from recent approaches in molecular genetics have allowed describing new subsets with potential therapeutic implications. Besides, the latter AML classification (“2017 European LeukemiaNet (ELN) risk stratification”) integrates both molecular alterations and conventional cytogenetics. High-resolution single-nucleotide polymorphism array (SNP-Array) identifies copy number alterations (CNA) and copy-neutral losses of heterozygosity (CN-LOH). In the randomized ALFA-0702 study (NCT 00932412), we have recently reported the effects of a CLARA combination (clofarabine + intermediate-dose cytarabine) as compared to standard high-dose cytarabine (HDAC) regimen as consolidation chemotherapy in younger adults with AML. Here, we aimed at evaluating the effects of the CLARA regimen according to patient subsets defined by SNP-array abnormalities.Methods. A total of 221 patients aged 18-59 years old with intermediate- or adverse-risk de novo AML in first complete remission (CR/CRp) after a common intensive induction course were randomized in the ALFA-0702 trial (Thomas et al. JCO 2017). The primary endpoint was relapse-free survival (RFS). Bone marrow or peripheral blood samples at diagnosis were analyzed by Cytoscan HD array (Affymetrix) according to the manufacturer's instructions. Data were screened according to the ChAS 3.1 algorithm, with stringent filter retaining CNA larger than 20 kb - 20 markers, telomeric CN-LOH > 3Mb and interstitial CN-LOH > 10Mb.Results. SNP-array was successfully performed in 187 out of the 221 randomized patients (92 CLARA arm, 95 HDAC arm). Twenty-three patients could not be analyzed in SNP-array for the following reasons: DNA sample was not available (15 patients) or in insufficient quantity (8 patients). SNP-array analysis failed in 11 patients. Median number of genomic SNP-Array abnormalities reached 2 per patient (range, 0-46) and did not significantly differ between both randomization arms (p=0.41). CNA accounted for the most common SNP-Array abnormalities (Loss=54%, Gain=38%, vs. CN-LOH=8%). Conventional cytogenetic analysis showed a complex karyotype (defined by at least 3 chromosomal abnormalities) in 37 patients (11 CLARA, 16 HDAC). In such patients, CLARA consolidation improved RFS (median RFS, 22 vs. 8 months; p=0.027). Interestingly, a similar impact was observed in the 56 patients (27 CLARA, 29 HDAC) with micro-complex karyotypes defined by 4 or more SNP-Array abnormalities. Notably, 7 patients with complex karyotype did not display micro-complex karyotype because of low bone marrow/blood blast infiltration and/or balanced alterations. In patients with micro-complex karyotypes, median RFS was 22 months in the CLARA arm compared to 10 months in the HDAC arm (p<.0001). The rate of patients who received allogeneic stem cell transplantation in first CR did not differ between the two arms (p=0.99). Importantly, CLARA consolidation tended to overcome the poor prognostic impact of patients with micro-complex karyotype. Indeed, patients with such lesions had similar 4-years RFS than other patients in the CLARA arm (44% vs. 47%) while it remained a poor prognosis factor in the HDAC arm (14% vs. 47%; Figure 1).Conclusion. As compared to conventional cytogenetics, SNP-array represents a high-resolution approach to better characterize molecular profile of AML patients by pointing out cryptic molecular abnormalities. Our results suggest that clofarabine-based consolidation benefits both patients with complex karyotypes and micro-complex karyotypes defined by 4 and more SNP-Array abnormalities. Therefore by delineating micro-complex karyotypes in SNP-array, we have defined a new subset of AML patients that could potentially benefit from a clofarabine-based consolidation regimen (21 additional patients in comparison with complex karyotypes). SNP-array could thus help to improve AML management by refining adverse patient subgroups that could potentially benefit from new alternative consolidation regimen. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Phase II Trial of Sirolimus with Standard Induction Chemotherapy in Patients with De Novo Acute Myeloid Leukemia

Background: The initial treatment of FLT3 wild type acute myeloid leukemia (AML) has not significantly changed since induction therapy with Ara-C and anthracyclines was first developed. Preclinical data suggests constitutive activation of the AKT3/mammalian target of rapamycin (mTOR) pathway may play a role in pathogenesis of this disease in a subset of AML patients. Previous data from our group has shown that the presence of phosphorylated ribosomal S6 (pS6) in AML blasts as detected by flow cytometry may predict response to the combination of sirolimus and induction chemotherapy. Here we report the clinical and pharmacodynamics results of a phase II study of the combination of these drugs.Methods: Subjects had newly diagnosed AML based on WHO criteria. Subjects received oral sirolimus starting on day 1 (12 mg loading dose) then 4 mg daily on days 2-10 with idarubicin 12mg/m2 days 4-7 and cytarabine 100mg/m2 days 4-10. Clinical response was assessed at hematologic recovery or by day 42 using IWG for response. Samples for pharmacodynamic studies of blasts were drawn prior to treatment with sirolimus on day 1, and then again prior to dosing of idarubicin and cytarabine on day 4. Assessment of pS6 on gated blasts, a biomarker of mTORC1 activation, was assessed by flow cytometry using previously described methods.Results: 55 patients enrolled on this trial received sirolimus with an average age of 58 years. Toxicity was similar to published 7+3 data and prolonged aplasia without recovery was not observed. Twenty-nine patients (53%) had high risk cytogenetic or molecular mutations, 15 (27%) had intermediate risk disease, and 11 (20%) patients had lower risk disease. As a whole, 35 patients (64%) entered into CR or CRp, 1 had a PR, 17 (27%) were non-responders. Two (4%) patients (ages 64 and 71) died during induction. Thirteen (45%) patients with high risk disease had a CR or CRp, 14 (48%) were non-responders, and 2 died in induction. Of the 15 subjects with intermediate risk disease, 11 (79%) patients had a CR or CRp, 1 had a partial response, and 3 were non-responders. All low risk patients had a CR, and 9 of 11 are alive and in remission at the time of censoring of data with a median time on study of 674 days. One patient with low risk disease subsequently relapsed, went to stem cell transplant, and is currently in CR. Two patients with low risk disease refused to proceed with standard of care consolidation; of these, 1 is alive and in remission 946 days from study entry, and 1 died in relapse. Fifteen patients (27%) subsequently underwent allogeneic stem cell transplantation in CR, and 8 (53%) are still alive an average of 358 days after transplant.Thirty-seven of 55 patients had adequate samples for pharmacodynamic analysis. Surprisingly, all patients demonstrated activation of ribosomal S6 prior to therapy in contrast to 67% positivity seen in previous studies that focused on patients with relapsed AML. mTORC1 was inhibited greater than 40% in 28/37 (76%) of patients. Inhibition did not correlate with either overall survival or age of patients.Conclusions: Sirolimus and 7+3 is a well-tolerated regimen. mTORC1 appears to be activated in almost all patients at the time of diagnosis of AML as assessed by flow cytometric assessment of ribosomal S6. Inhibition of mTORC1 did not correlate with response, suggesting that AML cells may have redundant signaling pathways that regulate chemosensitivity when mTORC1 is inhibited. Given the large percentage of samples with mTORC1 activation in this upfront AML study, future research into dual mTORC inhibitors may prove valuable. DisclosuresCarroll:Astellas Pharmaceuticals: Research Funding; Incyte Pharmaceuticals: Research Funding. Porcu:Innate Pharma: Research Funding; Kura: Research Funding; Miragen: Research Funding; Kiowa: Research Funding; Galderma: Research Funding; Tetralogic: Research Funding; Cell Medica: Research Funding; Celgene: Research Funding. Perl:Arog Pharmaceuticals: Consultancy; Novartis: Other: Advisory Board; Pfizer: Other: Advisory Board; Seattle Genetics: Other: Advisory board; Asana Biosciences: Other: Scientific advisory board; Astellas: Consultancy; Daiichi Sankyo: Consultancy; Actinium Pharmaceuticals: Other: Scientific Advisory Board.

Simultaneous Identification and Stratification of Low Molecular Risk AML Patients Using a Single LSC17-Based Nanostring Assay at Diagnosis

Refractoriness to induction chemotherapy and relapse after achievement of remission are the main barriers to cure in acute myeloid leukemia (AML). Currently, most AML patients receive standard induction chemotherapy because risk stratification depends on cytogenetic and molecular tests that typically take weeks to return results. We recently reported a rapid 17-gene leukemia stem cell score (LSC17, Ng et al., Nature 2016) that can accurately determine risk within 24 to 48 hours of diagnosis. Patients with high LSC17 scores have poor outcomes with current treatments and could be enrolled in clinical trials evaluating novel upfront treatment strategies.A small subset of AML patients with normal cytogenetics, NPM1 mutation and no FLT3-ITD are classified as low molecular risk (LMR) and are considered to have better outcomes, but ~35% relapse within 2 years following conventional therapy. In our prior study, we derived an optimized, re-weighted sub-score for LMR patients in which only 3 of the 17 genes contribute to the calculated score (LSC3). A high LSC3 score identifies LMR patients who have worse outcomes following standard induction therapy, and who might benefit from novel frontline therapy. Currently however, the LSC3 score can only be employed for risk-adapted therapy decisions in the post-remission setting, as LMR patients are only identified following cytogenetic and molecular testing. Earlier identification of high-risk LMR patients will facilitate clinical trials of novel frontline therapy in this patient subset.To determine whether a subset of the LSC17 signature genes could be used to identify LMR cases at diagnosis, we applied sparse binomial regression to relate the expression of the 17 signature genes to LMR vs. non-LMR cases in a large training cohort comprising AML patients of all subtypes (GSE6891, n=495 patients including 48 [9%] LMR cases). The resulting 13-gene sub-score (LMR13) can accurately identify LMR patients in 6 large independent validation cohorts spanning n=1559 cytogenetically and molecularly diverse AML patients using gene expression data from several different platforms (Figure 1; microarray datasets: GSE37642, n=542, area under the receiver operating characteristic curve (AUROC)=0.88; GSE15434, n=251, AUROC=0.92; TCGA AML, n=183, AUROC=0.92; ALFA-0701, n=192, AUROC=0.89; RNA-Seq dataset: TCGA AML, n=169, AUROC=0.92; NanoString datasets: GSE76004, n=237, AUROC=0.96; PM AML, n=154, AUROC=0.86). Importantly, the LMR13 score for each patient can be calculated from the same NanoString assay used to determine the patient's LSC17 and LSC3 risk scores. The ability to identify LMR cases rapidly at diagnosis enhances the clinical utility of the LSC17 assay, as it will allow clinicians to use the most appropriate risk score (LSC3 vs LSC17) for all newly-diagnosed AML patients, and enable evaluation of novel upfront treatment strategies for higher-risk LMR cases. Furthermore, integration of our functionally defined stem cell gene list with patient survival data using statistical learning approaches provides a basis for ongoing characterization of the molecular pathways associated with clinical outcomes, irrespective of mutational profile. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Study to Predict Response to Azacitidine or Lenalidomide/Azacitidine Combination Therapy in MDS and AML Patients Using a Computational Biological Modeling (CBM) Approach

Background: Several treatment options are available for patients with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML); however, the majority of patients will fail treatment or relapse. Predicting response is imperative in determining the right treatment regimen for each patient but currently no validated method exists.Aim: In this study, we aimed to predict treatment response in patients with MDS or AML using Cellworks' computational biology modeling (CBM) approach.Methods: In total, 16 patients were included in this analysis. 11 patients were treated on a single-institution phase II study of 5-day intravenous azacitidine (AZA) in patients with previously untreated MDS (Martin et al, AM J Hematol 2009 [NCT00384956]). An additional 5 patients were treated on a single-institution phase I study of daily high-dose lenalidomide (LEN) and 5-day intravenous AZA induction followed by low dose LEN and AZA maintenance in patients with newly diagnosed or relapsed AML (Ramsingh et al, Leukemia 2013 [NCT01016600]). Both treatments were generally well tolerated and showed promising response rates.For this analysis, whole exome sequencing (SureSelectXT Clinical Research Exome) and CGH microarrays were performed on previously collected samples associated with the respective clinical trials. The genomic data and non-genomic clinical data (age, diagnosis, white blood cell count, hemoglobin, platelet count, and bone marrow blast percentage) were used for CBM and response prediction. Digital drug simulations were conducted by quantitatively measuring drug effect on a cell growth score (proliferation + viability + apoptosis) along with impact on a identified disease specific biomarker unique to each patient. Each patient-specific protein network was screened for the extent by which AZA or LEN+AZA, respectively, reduced disease growth. The CBM tool used simulation prediction and clinical data through weighted decision tree analysis for the final response call. The predicted response was compared with the clinical response to assess for accuracy and predictive value. Patients who had a partial response or better per standard IMW criteria were classified as responders, all others were classified as non-responders.Results: 16 drug predictions were made by the CBM with 81% accuracy, 86% positive predictive value, 78% negative predictive value, 75% sensitivity and 88% specificity.In the subset of MDS patients treated with AZA, 4/11(36%) were classified as responders and 7/11 (64%) non-responders per IMW criteria. The median age at study entry was 73 and 6/11 were male. The median number of cycles of AZA administered was 6 (range 1-6). The CBM accurately predicted the clinical outcomes from 9/11 patients including 2/4 responders and 7/7 non-responders.In the subset of AML patients treated with LEN + AZA, 4/5 (80%) were classified as responders and 1/5 (20%) non-responders per IMW criteria. The median age at study entry was 71 and 1/5 were male. 3 of the patients had newly diagnosed AML and 2 had relapsed disease. The median number of cycles of LEN + AZA administered was 5 (range 1-17). The CBM accurately predicted the clinical outcomes from 4/5 patients including 4/4 responders.Conclusions: The CBM tested herein, which included genomic abnormalities and clinical data, showed high predictive value of protein network aberrations and clinical outcomes. The study validates the approach to a priori predict response and identify the right therapy option for the patient and could be used to establish eligibility criteria for precision enrollment in drug development trials. In addition, the network method uncovered possible molecular reasons for drug failure and resistance pathways that could be targeted to induce chemo sensitivity. This clinical decision support technology could enable improved effectiveness of therapy and help avoid unneeded toxicity and reduce health care expenses by eliminating unnecessary treatments. DisclosuresVij:Bristol-Meyers-Squibb: Honoraria; Takeda: Honoraria, Research Funding; Celgene: Honoraria; Abbvie: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria; Konypharma: Honoraria; Jazz: Honoraria. Singh:Cellworks Research India Pvt. Ltd: Employment. Sahu:Cellworks Research India Pvt. Ltd: Employment. Kumari:Cellworks Research India Pvt. Ltd: Employment. Narayan:Cellworks Research India Pvt. Ltd: Employment. G:Cellworks Research India Pvt. Ltd: Employment. Vali:Cellworks Group Inc.: Employment. Abbasi:Cellworks Group Inc.: Employment.

Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

SummaryThe aberrant expression of dopamine receptors (DRDs) in acute myeloid leukemia (AML) cells has encouraged the repurposing of DRD antagonists such as thioridazine (TDZ) as anti-leukemic agents. Here, we access patient cells from a Phase I dose escalation trial to resolve the cellular and molecular bases of response to TDZ, and we extend these findings to an additional independent cohort of AML patient samples tested preclinically. We reveal that in DRD2+ AML patients, DRD signaling in leukemic progenitors provides leukemia-exclusive networks of sensitivity that spare healthy hematopoiesis. AML progenitor cell suppression can be increased by the isolation of the positive enantiomer from the racemic TDZ mixture (TDZ+), and this is accompanied by reduced cardiac liability. Our study indicates that the development of DRD-directed therapies provides a targeting strategy for a subset of AML patients and potentially other cancers that acquire DRD expression upon transformation from healthy tissue.

Chemotherapy-Induced Survivin Regulation in Acute Myeloid Leukemia Cells

Survivin is a 16.5 kDa protein highly expressed in centrosomes, where it controls proper sister chromatid separation. In addition to its function in mitosis, survivin is also involved in apoptosis. Overexpression of survivin in many cancer types makes it a suitable target for cancer therapy. Western blotting and confocal microscopy were used to characterize the effect of chemotherapy on acute myeloid leukemia (AML) cells. We found enhanced survivin expression in a panel of AML cell lines treated with cytarabine (Ara-C), which is part of a first-line induction regimen for AML therapy. Simultaneously, Ara-C caused growth arrest and depletion of the mitotic cell fraction. Subsequently, the effect of a second component of standard therapy protocol, idarubicin, and of a known survivin inhibitor, YM-155, on cell viability and survivin expression and localization in AML cells was investigated. Idarubicin reversed Ara-C-induced survivin upregulation in the majority of AML cell lines. YM-155 caused survivin deregulation together with a viability decrease in cells resistant to idarubicin treatment, suggesting that YM-155 might be efficient in a specific subset of AML patients. Expression levels of other apoptosis-related proteins, in particular X-linked inhibitor of apoptosis (XIAP), Mcl-1, and p53, and of the cell-cycle inhibitor p21 considerably changed in almost all cases, confirming the off-target effects of YM-155.

A Pilot/Safety Study of sEphB4-Hsa in Combination with a Hypomethylating Agent for Patients with Relapsed or Refractory Myelodysplastic Syndrome and Acute Myeloid Leukemia Previously Treated with a Hypomethylating Agent

Background: Myelodysplastic syndrome (MDS) is a clonal hematopoietic neoplasm that results in bone marrow failure and frequently leads to acute myeloid leukemia (AML). Hypomethylating agents (HMA) are the only FDA-approved treatment for MDS and among few options for chemotherapy-ineligible patients with AML. There are limited options for patients in whom HMA therapy fails and who are not candidates for allogeneic stem cell transplant, so therapies that complement or restore sensitivity to HMAs are needed. Studies have shown increases in bone marrow microvessel density (MVD) and angiogenic markers in patients with MDS and AML. A decrease in MVD has been shown to correlate with response to hypomethylating agents in MDS. A receptor-ligand interaction, comprised of receptor EphB4 and membrane localized ligand EphrinB2, mediates angiogenesis in normal tissue and appears to be a target unique to many cancer types. We have previously shown EphB4 to be highly expressed and a driver of leukemic cell survival in a subset of AML patients. Our group has developed a human fusion protein, sEphB4-HSA, that blocks bidirectional signaling induced by EphB4-EphrinB2 interaction to inhibit tumor cell proliferation and angiogenesis. In phase I clinical trials of sEphB4-HSA in various tumor types, there were no myelosuppressive effects and minimal toxicity. Given its safety in phase I and potential to inhibit leukemic cell proliferation and angiogenesis, we proposed a pilot trial to evaluate the safety of sEphB4-HSA in combination with HMAs in MDS and AML patients who have failed treatment with HMAs. Methods: This pilot study was designed to enroll 6 patients with relapsed/refractory intermediate or high-risk MDS and 6 patients with AML refractory to or relapsed to HMA treatment and who are deemed unfit for chemotherapy. Treatment consisted of sEphB4-HSA 15 mg/kg IV every 2 weeks in combination with the FDA-approved HMA most recently or currently being used for treatment (decitabine 20mg/m2 IV/1hr on days 1 to 5 every 28 days or azacitidine 75mg/m2 SC or IV on days 1 to 7 every 28 days). Patients were treated for as long as they were receiving clinical benefit up to 12 months. The primary endpoint was toxicity and tolerability of sEphB4-HSA in combination with HMA. Toxicity was assessed and graded after each cycle according to the CTCAE version 4. Tolerability was defined as the ability to complete two cycles of treatment without the occurrence of dose-limiting toxicity. A secondary efficacy endpoint was to assess best overall response, based on the IWG Working Group Criteria for MDS and AML, during the first two cycles of treatment. Enrollment was stopped after 7 patients due to expiration of funding. Results: Three patients with intermediate-risk MDS were treated for a median duration of 6 cycles (2-12) and 4 patients with AML were treated for 2 cycles. Median age was 75.5 years (67.9-84.8) and 57.1% were male. HMA included azacitadine in 6 patients and decitabine in one patient. There were no dose-limiting toxicities. There were 8 grade 3/4 events attributed to HMA, which included: neutropenia (2), thrombocytopenia (3), and leukopenia (3). There were 3 grade 3/4 events attributed to sEphB4, which included: febrile neutropenia (1), leukopenia (1), and hypertension (1). Of the MDS patients, 2 had stable disease, 1 patient after 2 cycles and 1 patient after 4 cycles. One patient achieved a hematologic improvement-erythroid after 6 cycles. AML patients had no disease response. Reasons for treatment discontinuation were death (1), disease progression (2), patient's decision (1), physician's decision (2), and hospice (1). Notably, a comparison of bone marrow biopsies at baseline and after 8 weeks of treatment demonstrated a decrease in MVD (Figure 1). Discussion: This pilot study found sEphB4 in combination with HMAs to be tolerable with no significant toxicity beyond that expected with HMA therapy and associated with potential clinical benefit in MDS patients. Improvement in abnormal bone marrow MVD may indicate a potential for sEphB4-HSA plus HMA therapy to alter the malignant microenvironment in MDS/AML. Disclosures No relevant conflicts of interest to declare.

Investigation of ALLO-316: A Fratricide-Resistant Allogeneic CAR T Targeting CD70 As a Potential Therapy for the Treatment of AML

CD70, a member of the TNF superfamily, is a type II transmembrane glycoprotein that interacts with its receptor (CD27) to promote survival of primed T cells and leads to formation of effector and memory T cells. Expression of CD70 in normal tissues is restricted to activated T and B lymphocytes and mature dendritic cells. CD70 is also widely expressed in various malignancies, including renal cell carcinoma (RCC) and acute myeloid leukemia (AML). The restricted expression pattern of CD70 in normal tissues makes it an attractive target for cancer therapeutics. Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is an exciting new therapeutic modality showing great promise in hematologic malignancies. Approval of two CD19-targeting autologous CAR Ts, Kymriah® and Yescarta®, has been followed with promising results from BCMA autologous CAR T clinical trials, showing that activity can extend to other targets. We have previously described the functional screening of a library of anti-CD70 scFv-based CARs and the identification of lead CD70 allogeneic CAR T cells (AlloCAR TTM) with robust activity against RCC cell lines both in vitro and in vivo. Here, we evaluate the anti-tumor activity and safety of a lead CD70 AlloCAR T (ALLO-316) for the treatment of AML. CD70 expression was evaluated and detected on three AML cell lines and in six primary AML patient samples, with 5/6 patient samples showing expression on 24%-99% of cells. CD70 expression will be profiled in a broader subset of AML patients and preliminary data will be presented. Despite the expression of CD70 on activated T cells it was possible to generate CD70 AlloCAR T cells. No CD70 expression was observed on CAR T cells after generation, suggesting either cells are succumbing to fratricide or are being "masked" by the CAR. CD70 was also not detected on Jurkat cells expressing CARs and this data, in combination with results showing CAR expression is protective when overexpressed in RCC cells support the phenomenon of "masking". Cellectis' TALEN® gene-editing was used to inactivate the TRAC and CD52 loci with the intent to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. ALLO-316 cells were highly effective at lysing CD70-expressing target cells and eliminated greater than 99% of cells at the high effector to target (E:T) ratio and were unable to lyse AML cells in which CD70 was knocked out. Moreover, ALLO-316 cells were able to kill primary AML blasts with CD70 expression ex vivo. An orthotopic in vivo model utilizing the AML cell line MV4-11 was developed and anti-tumor activity was observed. In addition to evaluating efficacy against AML cell lines and tumors we also explored potential toxicity liabilities related to ALLO-316 treatment. Previous studies have reported that certain AML tumor antigens can also be expressed on normal hematopoietic progenitors and such expression could potentially lead to toxicity with targeted therapeutics. No detectable CD70 was observed by flow cytometry on purified CD34+ cells from 14 healthy donors. Taken together, our results support clinical development of CD70 AlloCAR T therapy for the treatment of AML. Disclosures Srinivasan: Allogene Therapeutics: Current Employment. Tan:Allogene Therapeutics: Current Employment. Cheng:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang:Allogene Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tacheva-Grigorova:Allogene Therapeutics: Current Employment, Current equity holder in publicly-traded company. Van Blarcom:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sommer:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sasu:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Panowski:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company.

Abstract IA18: The PI3K isoforms in myeloid leukemia and hematopoietic stem cells

Abstract The Class IA PI3 kinase isoforms (p110α, β, and δ) transduce many growth factor signals that are important for the proliferation, differentiation, and self-renewal of hematopoietic stem cells (HSCs). Mutations in growth factor receptors or RAS proteins are commonly observed in patients with acute myeloid leukemia (AML), leading to activation of the PI3K/AKT pathway. Therefore, PI3K inhibition is an attractive therapeutic strategy for a large subset of AML patients. We previously reported that p110α is dispensable for HSC function, suggesting that redundancy exists between the Class IA isoforms in HSCs (Gritsman et al., J Clin Invest 2014). However, we have identified a specific role for p110α in RAS-mutated myeloid leukemia. Furthermore, we found that the p110α-selective inhibitor BYL-719 can sensitize RAS-mutated leukemic cells to the MEK inhibitor MEK-162. While PI3K inhibitors have multiple clinical indications, including in hematologic malignancies, it is still unclear whether PI3K plays an important role in normal HSC function. Upon environmental stresses like chemotherapy or infection, quiescent HSCs must enter the cell cycle to re-establish homeostasis and promote emergency myelopoiesis. To test for redundancy between p110α and p110δ in HSCs, we generated p110α and p110δ double knockout (DKO) mice, with germline deletion of p110δ and conditional deletion of p110α. DKO mice have leukopenia, anemia, and decreased numbers of lymphoid-primed multipotent progenitors (LMPPs) in the bone marrow, though HSC numbers are unchanged. In competitive repopulation assays, B-cell maintenance was severely impaired, while the myeloid and T-cell lineages were relatively preserved. This suggests that HSC function is not impaired at steady state. We performed transcriptome analysis of DKO, p110δ, and p110α KO HSCs and LMPPs after bone marrow transplantation. Gene set enrichment analysis revealed downregulation of gene sets associated with cell cycle progression. Interestingly, we also observed negative enrichment of inflammatory response gene sets in both p110δ KO and DKO HSCs and LMPPs. To examine the roles of p110δ and α in the hematopoietic stress response, we injected p110δ, DKO, and WT mice with 5-fluorouracil. We observed significantly decreased survival of 5-FU-treated DKO mice due to impaired hematopoietic recovery, associated with the failure of HSCs to enter the cell cycle. Consistent with this finding, we also observed a decrease in myeloid reconstitution by DKO HSCs in secondary competitive transplantation, suggesting that p110α and p110δ play redundant roles in emergency myelopoiesis. We also observed defects in downstream signaling in DKO HSCs and progenitors in response to IL1β or TNFα stimulation. Our results suggest that p110α and δ act in a redundant fashion to transduce specific inflammatory signals in HSPCs in response to hematopoietic stress. Our results have important implications for the use of PI3K inhibitors in combination with chemotherapy and in other hematopoietic stress conditions. Citation Format: Shayda Hemmati, Taneisha Sinclair, Meng Tong, Boris Bartholdy, Rachel Okabe, Kristina Ames, Leanne Ostrodka, Tamanna Haque, Imit Kaur, Anupriya Agarwal, Jean Zhao, Thomas Roberts, Kira Gritsman. The PI3K isoforms in myeloid leukemia and hematopoietic stem cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA18.

Abstract 1921: The DDB2 intronic variation rs830083 is a strong predictor of gemtuzumab ozogamicin response in AML patients: A calicheamicin pathway-directed analysis from the COG-AAML0531 trial

Abstract Polymorphisms in DNA damage repair (DDR) genes could serve as predictors of response to gemtuzumab ozogamicin (GO), a CD33 targeting antibody conjugated to the DNA damaging agent calicheamicin approved for use in immunotherapy for acute myeloid leukemia (AML). GO is internalized upon binding to the myeloid cell surface antigen CD33, allowing calicheamicin-mediated DNA damage and subsequent apoptosis. Following concerns over safety, GO was withdrawn in 2010 but was re-approved in 2017 with the emergence of new evidence of its clinical efficacy and safety. In clinical trials, significantly improved event free survival (EFS), disease free survival (DFS), and relapse risk (RR) were observed in a subset of AML patients with the addition of GO to conventional AML chemotherapy. Combined with low overall survival (OS) and high RR with the relatively unchanged conventional AML chemotherapy, GO usage is anticipated to increase. We used samples from the Children's Oncology Group (COG) trial AAML0531 to determine the association between polymorphism in DDR genes and OS, EFS, DFS, and RR with GO treatment as the primary end points. In this trial, newly diagnosed AML patients were randomized to receive either the standard 5 course chemotherapy alone (Arm A) or with two doses of GO (3mg/m2/dose) during induction 1 and intensification 2 (Arm B). Briefly, genomic DNA samples from a total of 947 patients from Arm A and Arm B were genotyped for 131 SNPs across 43 key genes involved in calicheamicin-mediated DDR. Overall, 28 SNPs in 21 genes were significantly associated with OS, EFS, DFS, and RR. Of these, 21 SNPs in 16 genes were associated with response to GO treatment (Arm B; p ≤ 0.05) and not with conventional AML chemotherapy (Arm A; p ≥ 0.05). The top 5 SNPs occurred within DDB2, XPA, XPC, and XRCC1 – genes involved in the nucleotide excision repair (NER) DDR pathway. The intronic variation rs830083 (G&amp;gt;C) in DDB2 had the poorest outcome (OS Cox p = 0.01; EFS cox p = 0.004; DFS p = 0.000; RR reg p = 0.0006). Furthermore, rs830083 was also most significantly associated with standard risk patients (DFS p = 0.003), EFS (p=0.01) and RR (p = 0.0006). DDB2 has been reported to play a critical role in apoptotic processes post-DNA damage induction by regulating p53, lending further importance to the association between rs830083 and poor response to treatment. In conclusion, the identification of several variants associated with treatment outcomes in our study underscores the importance of a pathway-directed SNP-based candidate gene approach to identify personalized approaches for AML treatment. Specifically, screening DDB2 SNPs could be applied in patient selection and predicting response to GO treatment. Citation Format: Vivek M. Shastri, Lata Chauhan, Todd A. Alonzo, Yi-Cheng Wang, Richard Aplenc, Betsy A. Hirsch, Alan S. Gamis, Soheil Meshinchi, Jatinder K. Lamba. The DDB2 intronic variation rs830083 is a strong predictor of gemtuzumab ozogamicin response in AML patients: A calicheamicin pathway-directed analysis from the COG-AAML0531 trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1921.