Subtype Of Acute Myeloid Leukemia Research Articles

Activation of GCN2 By HC-7366 Results in Significant Anti-Tumor Efficacy As Monotherapy and Overcomes Resistance Mechanisms When Combined with Venetoclax in AML

Venetoclax (anti-BCL2) is approved for elderly patients with acute myeloid leukemia (AML) in combination with hypomethylating agents such as azacitidine or decitabine. However, several resistance mechanisms arise, impeding responses in patients. These resistance mechanisms include TP53 or FLT3-ITD mutations, suppression of pro-apoptotic proteins NOXA or PUMA, increases in oxidative phosphorylation and energy metabolism, or elevated expression of S100A8/A9. Recent reports have highlighted that hyperactivation of the integrated stress response (ISR) pathway overcomes venetoclax resistance through NOXA activation and glycolytic activity inhibition. However, these studies used drugs that indirectly activate the ISR with other off-target effects. General Control Nonderepressible 2 (GCN2) is an ISR kinase that senses and responds to nutrient stress conditions, and activation of GCN2 results in antitumor activity. We are developing HC-7366, a first-in-class, first-in-human direct GCN2 activator, and are currently evaluating this agent in a phase 1 clinical trial in solid tumors (NCT05121948). Here we present a series of studies characterizing the antitumor effects of HC-7366 in AML, and how it impacts venetoclax resistance. AML patients with TP53 mutations are known to have lower response rates to venetoclax.The in vivo efficacy of HC-7366 in both CDX and PDX models was independent of TP53 mutation, resulting in 100% complete response and 100% tumor growth inhibition in TP53-mutated MOLM-16 and KG-1 tumor models, respectively. Analysis of tumors from treated mice by IHC demonstrated activation of the ISR as evidenced by increased expression of the ATF4 targets ASNS and PSAT1. A genome-wide CRISPR screen in KG-1 cells revealed that GCN2 sgRNAs were significantly enriched in HC-7366-treated cells, highlighting the importance of GCN2 activation in mediating the mechanism of action of HC-7366. Interestingly, BCL2 sgRNAs were significantly depleted in HC-7366 treated cells, suggesting a synthetic lethal relationship with BCL2 inhibitors, such as venetoclax, when combined with HC-7366. Indeed, in the MV4-11 FLT3-ITD mutant model (a differentiated subtype of AML that shows limited response to venetoclax), the combination of HC-7366 and venetoclax produced substantial benefit resulting in 26% tumor regression and enhanced activation of the ISR pathway. HC-7366 treatment also increased mRNA and protein levels of NOXA and PUMA while reducing mitochondrial respiration and glycolysis in a GCN2-dependent manner, leading to a low energetic state. Consistent with its impact on cellular energetics, global metabolomic analyses of AML xenograft tumors showed that HC-7366 significantly altered metabolites associated with glycolysis and the TCA cycle. Additionally, HC-7366 reduced protein levels of S100A8/A9, also known to correlate with venetoclax resistance. Our in vitro and in vivo results demonstrate that HC-7366 is a potent GCN2 activator with strong antitumor activity in AML as a single agent and in combination with venetoclax. The potential of HC-7366 to counteract multiple known resistance mechanisms to venetoclax could provide a viable treatment option for patients with relapsed/refractory AML when used in combination with venetoclax.

RTK-RAS Signaling Pathway Was Enriched in Rare Acute Myeloid Leukemia Patients with t(16;21)(p11;q22)/ FUS::ERG

Introduction: t(16;21)(p11;q22)/ FUS::ERG is a rare recurrent abnormality in acute myeloid leukemia (AML) and confers a poor prognosis in AML patients. Previous studies analyzed the role of allogeneic hematopoietic stem cell transplantation (HSCT) in these patients but the results were discordant. AML is characterized by concurrent mutations, which cooperated in the leukemogenesis and refractory/relapse mechanism. However, few studies described the mutational landscape of FUS::ERG AML. Olshanskaya et al. discovered concomitant DNMT3A, ASXL1, RUNX1 and BCOR mutations in the FUS::ERG AML patients but the study was limited to the small case number. Therefore, we aim to assess genomic alterations and clinical outcome of FUS::ERG AML patients with a larger cohort. Methods: We reviewed medical records of 12328 AML patients diagnosed between December, 2011 and February, 2022 in our hospital. A total of 39 consecutive AML patients with karyotype or reverse transcription-polymerase chain reaction analysis proven t(16;21)(p11;q22)/ FUS::ERG were identified. Whole exome sequencing was performed in 4 patients and targeted sequencing was performed in 20 patients. Patients with follow-up time longer than 30 days were included in the survival analysis. Overall survival (OS) was calculated from the date of diagnosis to the date of death or censoring. Event free survival (EFS) was calculated from the date of diagnosis to the date of refractory, relapse, death or censoring, whichever came first. Results: M5 was the predominant subtype according to French-American-British classification systems. CD56 (91.9%) and CD123 (81.1%) were found to be expressed in most FUS::ERG AML patients. Six patients had central nervous system involvement diagnosed by flowcytometry (FCM) analysis of cerebrospinal fluid at the time of first lumbar puncture. One patient was diagnosed central nervous system involvement at relapse (Table 1). The most prevalent somatic variants of our FUS::ERG AML patients were PTPN11 (25%), NRAS (20%), RELN (15%), CENPB (10%), RUNX1 (10%) and SH2B3 (10%) mutations (Figure 1). Five patients had no somatic mutation detected by targeted sequencing. Notably, RTK-RAS signaling pathway was the most involved pathway after clustering the aberrated genes according to the Gene Ontology terms, which included PTPN11, NRAS, ABL1 and JAK2 aberrations. All but one PTPN11 mutations affected the amino terminal (N)-SH2 domain. NRAS mutation were found mutated at amino acid G12 and Q61. The median EFS of the patients was 9.3 months (95% CI: 9.2-NA) and OS was 18.2 months (95%CI: 13.1-NA). Due to the poor outcome of this subtype of AML, allogeneic HSCT was highly recommended. A total of 14 patients underwent allogeneic HSCT. Notably, seven of ten patients were positive for minimal residual disease assessed by FCM at the time of transplantation. We did not observe the statistically significant effect of HSCT on improving the EFS (p = 0.24) or OS (p=0.26) of our patients. Conclusions: PTPN11 and NRAS were the most frequent mutations and RTK-RAS signaling pathway was the most involved pathway in rare AML patients with t(16;21)(p11;q22)/ FUS::ERG. The addition of signaling pathway inhibitors followed by HSCT might be an effective strategy to overcome the dismal outcome of this subtype. Larger cohort studies are warranted to investigate the molecular characteristics further as well as evaluate the clinical activity of the SHP2 (PTPN11) inhibitors in FUS::ERG AML patients.

Epigenetic Modulation Enhances the Therapeutic Potential of All-Trans Retinoic Acid in Acute Myeloid Leukemia

Epigenetic dysregulation has been strongly associated with the development and progression of acute myeloid leukemia (AML). While all-trans retinoic acid (ATRA), a derivative of vitamin A, has demonstrated significant anti-cancer effects in acute promyelocytic leukemia (APL-AML), its clinical efficacy in non-APL AMLs has been limited. This limited response can be attributed, in part, to the epigenetic silencing of genes targeted by or involved in the ATRA pathway, rendering them unresponsive to the drug. Consequently, our research aims to overcome the epigenetic barriers in AML by combining ATRA with small molecules that possess epigenetic modulating properties. To investigate the activation of the retinoic acid pathway in response to ATRA and a library of over 650 small molecules with epigenetic activity, we transduced HEK-293 cells with the p-GreenFire-RARE-Tk-Luc reporter construct. Through the screening process, we have identified 11 compounds that show promise as potential candidates for combination therapy with ATRA in non-APL AML. Notably, when combined with ATRA, two specific compounds, a dual PI3K HDAC inhibitor, and a pan-PKC inhibitor, demonstrated a significant 4-5 fold increase in RARE activity, indicating a synergistic effect. To determine the impact of selected compounds on the functional level, we evaluated the viability, differentiation, and phosphorylation status of ERK1/2, AKT1, MAPK9, p38, and MAPK9 in various cell lines, including HL-60, KG1a, BMNC, and primary MSC and AML cells. The dual PI3K HDAC inhibitor and a pan-PKC inhibitor were noncytotoxic to HL60, BMNC, and primary MSC. Interestingly, dual PI3K HDAC inhibitor with and without ATRA reduced the viability of KG1α by more than 95%. Moreover, dual PI3K HDAC inhibitor and a pan-PKC inhibitor, when combined with ATRA, lead to a 2-3 fold increase in the differentiation of HL60 cells and a 2-4 fold increase in the differentiation of AML cells ex vivo compared to ATRA in combination with the LSD1 inhibitor TCP (treatment control protocol) and untreated cells from AML subtypes M1, M2, and M4. PI3K HDAC inhibitor combined with ATRA significantly reduced p-MAPK and p-p38 levels while increasing the phosphorylation of p53 in HL-60 cells. Furthermore, RNA sequencing and chromatin profiling by Cleavage Under Targets and Tagmentation (CUT&Tag) were performed to gain additional insights into the molecular mechanisms involved. Based on obtained results, the combination of ATRA with specific dual PI3K HDAC and pan-PKC inhibitors holds promise as a potential therapeutic approach for AML. Nevertheless, further investigations, including studies utilizing patient-derived xenograft models, are warranted to validate the efficacy and feasibility of this proposed treatment strategy.

Is Allogeneic Hematopoietic Stem Cell Transplant (HCT) in First Remission Beneficial to Acute Myeloid Leukemia (AML) Patients with Isocitrate Dehydrogenase(IDH) Mutations?

Introduction Somatic mutations in the isocitrate dehydrogenase 1 and 2 genes (IDH1 and IDH2) are relatively frequent in acute myeloid leukemia (AML) but with heterogeneous prognoses. The prognostic impact of this AML subtype with IDH mutations may be influenced by the comutational status with other somatic mutation, the specific types of IDH1/2 mutation (ie, IDH1 R132, IDH2 R140, and IDH2 R172). Allogeneic stem cell transplant allogeneic HCT represents a curative option for subgroups of AML patients (pts), but not for all subtypes. Transplant referral is made based on the ELN risk group classified into favorable, intermediate, and adverse risk. Although many AML pts with IDH1/2 mutations are referred to allogeneic hematopoietic stem cell transplantation (HCT) and transplanted, however it is not fully elucidated whether allogeneic HCT brings definite survival benefit to this group of the pts. Statistical attention is to be paid to the time to HCT from CR1 achievement. Before HCT, the case should be counted as a case of no HCT, while after HCT this case can be accounted as the case received HCT using the Mantel-Byar test (MBT). To handle immortal time bias, the Mantel-Byar method was used to depict outcomes during the follow-up period. Kaplan-Meier curves were avoided as they can introduce immortal bias, considering the period of time before treatment as part of the treatment arm. Instead, the MBT and landmark methods were employed to address this issue. Allogeneic HCT was treated as a time-dependent variable to prevent immortal bias. The aim of the present study is to identify if AML pts with IDH mutations obtain survival benefit from allogeneic HCT in CR1 using HCT as a time-dependent covariate with using Mantel-Byar test. Patients & Method We conducted a retrospective study that included 1,228 adult pts who were diagnosed with AML and achieved CR1 in 5 centers (Toronto, Vancouver, and Montreal, Canada; Hwasun and Seoul, South Korea) from January 1997 to January 2020. Next-generation sequencing was performed in each institution according to their own standard targeted sequencing panel. We considered allogeneic HCT in CR1 as a time-dependent covariate and used a Mantel-Byar test to avoid immortal bias. Relapse-free survival (RFS) was selected for the primary endpoint, defined as the time from CR1 to relapse or death from any cause. EZR software version 1.41 was used throughout the study for statistical analysis. Results Out of 1228, a total of 238 pts (19.4%) was found to have IDH1/2 mutation, among whom 92 pts (38.6%) had isolated IDH1 mutations, 140 pts (58.8%), isolated IDH2 mutations, and 6 pts (2.5%) having coexistence of IDH1 and IDH2 mutations. With a median follow-up duration of 20.8 months (mo) (1-194.3 mo) among survivors after achieving CR1, 91 pts died while 115 pts experienced relapse. One hundred twenty-one pts received HCT in CR1, and 44 pts relapsed after HCT. The 2-year overall survival of those 238 pts was 58.9% (51.3-65.6), and 2-yr RFS was48.9% (41.6-55.8) Between the HCT (n=121) vs non-HCT group (n=117), there were no differences in sex, cytogenetic risk or other somatic mutation except NPM1 mutation which was more frequently detected in the non-HCT group (p =0.002). As expected, the HCT group was younger (55 years (15-74 yrs)) than non-HCT group (62 years (27-82 yrs), p<0.001). First, the RFS and OS was compared between HCT vs non-HCT groups considering HCT as non-time dependent covariate. The 2-year RFS rate was higher in the HCT group (61.0%) compared to the non-HCT group (37.0%, p= 0.0002). However, it was not translated into superior OS in the HCT group: 2-year OS rate, 64.4% in the HCT group vs 53.7% in the non-HCT group, p=0.1). We have conducted time-dependent analysis taking taken HCT in CR1 into account as a time-dependent (td) covariate using MBT. The trend of superior RFS in HCT group was reproduced in MBT: HCT in CR1 was found to improve RFS (Hazard ratio [(HR) of 0.62, (95% C.I. [0.42-0.92]; p=0.02)] as shown in Fig.1B utilizing Simon-Makuch plot. However, Multivariate analysis (MVA) for RFS did not confirm HCT in CR1 as an independent prognostic factor for RFS, while Following factors were found to be associated with RFS adversely: age > 60 years, presence of DNMT3A mutation, and presence of IDH1 mutation. Conclusion Allogeneic HCT in CR1 could bring some survival benefit on RFS in AML pts with IDH1/2 mutation, but not confirmed in MVA. Further study is warranted to reach a clearer conclusion with larger number of

High Throughput Microfluidics Platform to Assess Synthetic Lethality and Novel Therapeutic Drug Combinations

Molecularly targeted therapies have reshaped cancer treatment in recent decades; however, not all patient cancers are eligible for or effectively treated by such approaches. In addition, resistance mechanisms (both pre-existing and acquired) limit clinical benefit. To these ends, combination therapies involving targeted agents are increasingly being developed for their potential to induce potent synthetic lethalities, bypass drug resistance, promote durable responses, limit adverse side effects via reduced dosing, and expand treatment options. In the context of acute myeloid leukemia (AML) specifically, several efficacious combination therapies have been approved for specific patient subsets, such as all-trans retinoic acid (ATRA) plus arsenic trioxide in the PML-RARA fusion acute promyelocytic subtype and Midostaurin plus Cytarabine and Daunorubicin in FLT3-mutant AML. Despite the availability of these and other therapies, there remains an unmet need for treatments against AML subtypes lacking actionable driver alterations or for which current therapies are insufficient. A major hurdle limiting novel combination therapy discovery is the sheer combinatorics associated with mixing two or more agents (e.g., screening all possible pairs of 100 drugs in a single cell line requires nearly 5,000 combination experiments). Thus, there is a practical need for methodologies that prioritize test compounds among broad candidate sets and high throughput technologies for rapid and efficient screening. We have developed a combination drug screening workflow that integrates machine learning-based predictions of potentially synergistic compound pairs with a droplet microfluidics-based screening platform. Our physical platform, termed the FlowMatrix, contains a series of nano wells arrayed in a 96 x 96 grid (for 9,216 total wells) in which cells are incubated and compounds delivered via emulsified droplets. To facilitate drug combination screening, the FlowMatrix is loaded column-wise with a 96-element library and subsequently loaded a second time along its rows with either the same or an independent drug library. In our current setup, one FlowMatrix can capture complete data for up to 100 unique drug pairs, which includes control, single-agent, and 4x4 dose combination measurements for each pair. Following multi-day incubation, viability for each well is assessed by the number of live cells imaged with fluorescence microscopy. Combinations with high scoring synergy based on Bliss independence criteria are then pushed to validation and further characterization pipelines. We are currently focused on identifying novel combination therapies for AML. Using small molecule libraries of diverse compound classes and machine learning to prioritize initial screening candidates, we have to-date profiled >8,500 AML cell line-drug combinations in our system. This encompasses >2,900 unique drug combinations profiled in 7 distinct AML cell lines. Among ~270 hits that emerged from these screens are established clinical and preclinical AML combination therapies, in addition to candidates that, to the best of our knowledge, are novel in the context of AML. Strong established hits in our screen include ATR inhibition plus Gemcitabine treatment as well as several combinations involving the BCL-2 inhibitor Venetoclax with chemotherapies (Decitabine and Daunorubicin), Quizartinib, Idasanutlin, and mTOR inhibitors. Thus, we have developed an efficient and cost-effective high throughput drug combinations profiling system that has uncovered candidate therapies that may expand treatment options for patients afflicted by AML.

KDM6A Modulates Anti-Tumor Immune Response By Integrating Immunogenic Cell Death in Human Acute Myeloid Leukemia

Identifying tumor cell-epigenetic regulation of immune activation in acute myeloid leukemia (AML) would be instrumental in developing next-generation of targeted therapy. AML cells have low mutational burden; inefficient ‘cross-presentation’ through conventional dendritic cells (DC) and immune activation are the major challenges for AML immunomodulation. ‘Immunogenic cell death’ (ICD) represents changes in the composition of the tumor cell surface as well as the release of ‘damage associated molecular patterns’ (DAMPs) in the tumor microenvironment that stimulates the dysfunctional immune system. DAMPs in turn operate on a series of receptors expressed by DCs and natural killer (NK) cells and contribute to long-lasting, protective anti-tumor immunity. For example, plasma membrane exposure of endoplasmic-reticulum (ER)-translocated calreticulin (ecto-CRT), which functions as an ‘eat me’ signal, is an important hallmark of ICD. Previous study suggested that calreticulin (CALR) promotes immunity and type I interferon-dependent survival in mice with AML (Fucikova, Blood 2016; Chen, Oncoimmunology 2017). In addition, ecto-CRT on malignant blasts correlated with improved NK cell-mediated cytotoxicity in AML patients with superior overall survival (Truxova, Haematologica 2020). Ecto-CRT has also been identified as an endogenous sterile ligand for NK cells (Sen Santara, Nature 2023). Nevertheless, molecular regulation of ICD in the context of epigenetic derangements and inflammation and its implication in AML immunotherapeutic modulation is poorly understood. KDM6A is a histone 3 lysine 27 demethylase that plays tumor suppressor function in AML (Gozdecka, Nat Genet 2018; Sera, Blood 2021). KDM6A escapes X-chromosome inactivation, and loss-of-function deletion or point mutations associate with resistance to ‘3+7‘ chemotherapy (Stief, Leukemia 2020). We have identified that in comparison to control AML cells, deficiency of KDM6A ( KDM6A-kd) associated with daunorubicin/etoposide-induced ICD, which was manifested by significant increase (2-3 fold , P<0.05) in ecto-CRT, plasma membrane exposure of pE1F2α, HSP70 and HSP90 with extracellular release of HMGB1 and ATP. Similarly, pharmacological inhibition of JmjC catalytic function using GSK-J4 sensitized human AML lines as well as primary AML patient bone marrow-derived CD34 + hematopoietic stem/progenitor cells to ICD. Induction in ICD was accompanied by an elevated type I interferon production that was functionally active in promoting interferon regulated gene expression. Interestingly, treatment with acute recombinant IFN-β caused an increase in activated STAT1 and inflammatory gene expression in KDM6A-kd AML cells compared to control AML. Corroborating these results, gene set enrichment analysis revealed a significant enrichment (NES=1.708, P=0.007) of upregulated type I interferon signatures (n=40) in Kdm6a -/- primary leukemia cells. Re-analysis of ChIP-seq results indicated 1647 Kdm6a occupied genes, which were upregulated upon Kdm6a loss. Gene ontology (GO) analysis of the 1647 genes further suggested an enrichment ( P<0.001) of type I interferon-responsive GO terms, indicating Kdm6a role in interferon signaling. Ecto-CRT level relies on premortem ER stress. In OHSU AML, KDM6A expression correlates with ER stress/unfolded protein response. Mechanistically, quantitative-ChIP and promoter-reporter assays indicated that KDM6A-kd de-repressed ICD induced expression of ATF4 and HSPA5 through chromatin and transcriptional remodeling. In agreement with these findings, AML blasts-healthy immune cells ex vivo co-culture experiments revealed that increase in ICD in KDM6A-kd AML was associated with activation (~2-foldincrease inCD80 +CD86 +, P<0.05) and phagocytic function of DCs. Loss of KDM6A was shown to bestow ‘BRCAness’ (Boila, Leukemia 2023), and PARP inhibition itself can cause ICD and augment tumor immunity. We observed that there was a significant induction in ICD and expression of NKG2D ligands ULBP1, ULBP5 in KDM6A-kd AML cells treated with olaparib. In addition, olaparib and GSK-J4 treatment showed additive effects in ICD and immune activation using primary AML CD34 + cells. Together, we illustrate that KDM6A regulates ICD through epigenetic mechanisms, while KDM6A deficient AML subtypes could be sensitized with immunomodulatory targeted therapy.

Clinical Characteristics and Prognosis of Acute Myeloid Leukemia Patients with Protein Tyrosine Phosphatase Non-Receptor Type 11 Gene Mutation

Objective: The purpose of our study was to investigate the clinical characteristics, molecular biological characteristics and prognosis of patients with acute myeloid leukemia (AML) harboring protein tyrosine phosphatase non-receptor type 11 (PTPN11) gene mutation. Methods: The clinical data of 30 newly diagnosed adult AML patients with PTPN11 gene mutation were analyzed retrospectively. Prognostic analysis and screening of prognostic factors were conducted using the Kaplan-Meier method and Cox proportional hazards regression model. Results: High frequency mutation sites of PTPN11 gene were located in exon 3 of chromosome 12, which are D61 and A72（16.7%）, followed by E76 (13.3%). The median variant allele frequency (VAF) of PTPN11 mutant gene was determined to be 18.4%. The patients were divided into two groups according to PTPN11 VAF 35.3% (upper quartile). We observed that the peripheral blood leukocyte count in patients with VAF ≥35.3% was significantly higher than patients with VAF < 35.3% (p=0.019).Moreover, the higher VAF was closely related to M5 subtype AML (p=0.016) and internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3) (FLT3-ITD) mutation (p= 0.048). Taking PTPN11 VAF 20% and 35.3% as the cutoff value, the patients were divided into two groups, and the overall survival(OS) and event free survival (EFS) of these two groups were not significant. Multivariate analysis using the COX proportional hazards model identified that white blood cell count and Eastern Cooperative Oncology Group (ECOG) physical status score were independent risk factors affecting the prognosis of EFS. Conclusion: Our study suggests that PTPN11 VAF may not serve as a prognostic factor in patients with PTPN11 mut AML. However, newly diagnosed patients with high white blood cell count and poor performance status were identified as independent risk factors for EFS in PTPN11 mut AML. Keywords: Acute myeloid leukemia, PTPN11 mutation, clinical characteristics

Acute Myeloid Leukemia: Selecting Induction Therapy Based on Biological Disease Factors

Greater insight into the role of biologic disease factors in acute myeloid leukemia (AML) has led to more effective and personalized treatment options. Data presented at the NCCN 2023 Annual Congress: Hematologic Malignancies reflected the rapidly evolving treatment landscape, showcasing the significance of combination therapies and precision medicine in managing different AML subtypes. For patients deemed fit for induction with favorable-risk cytogenetics, the standard of care has moved to combining the 7 + 3 regimen with gemtuzumab ozogamicin. Alternatively, for patients with FLT3-mutated disease, a more intensified approach involving the addition of midostaurin or newer therapies, such as quizartinib, to standard induction treatment has shown improved outcomes. For patients with AML who are unfit for standard induction, treatments combining hypomethylating agents with venetoclax, as well as therapies involving IDH1/2 and FLT3 inhibitors for specific genetic subtypes, offer encouraging alternatives in managing the disease.

Genomic DNA-based measurable residual disease monitoring in pediatric acute myeloid leukemia: unselected consecutive cohort study

Measurable residual disease (MRD) monitoring in childhood acute myeloid leukemia (AML) is used to assess response to treatment and for early detection of imminent relapse. In childhood AML, MRD is typically evaluated using flow cytometry, or by quantitative detection of leukemia-specific aberrations at the mRNA level. Both methods, however, have significant limitations. Recently, we demonstrated the feasibility of MRD monitoring in selected subgroups of AML at the genomic DNA (gDNA) level. To evaluate the potential of gDNA-based MRD monitoring across all AML subtypes, we conducted a comprehensive analysis involving 133 consecutively diagnosed children. Integrating next-generation sequencing into the diagnostic process, we identified (presumed) primary genetic aberrations suitable as MRD targets in 97% of patients. We developed patient-specific quantification assays and monitored MRD in 122 children. The gDNA-based MRD monitoring via quantification of primary aberrations with a sensitivity of at least 10−4 was possible in 86% of patients; via quantification with sensitivity of 5 × 10−4, of secondary aberrations, or at the mRNA level in an additional 8%. Importantly, gDNA-based MRD exhibited independent prognostic value at early time-points in patients stratified to intermediate-/high-risk treatment arms. Our study demonstrates the broad applicability, feasibility, and clinical significance of gDNA-based MRD monitoring in childhood AML.

Reversal of MYB-dependent suppression of MAFB expression overrides leukaemia phenotype in MLL-rearranged AML

The transcription factor MYB plays a pivotal role in haematopoietic homoeostasis and its aberrant expression is involved in the genesis and maintenance of acute myeloid leukaemia (AML). We have previously demonstrated that not all AML subtypes display the same dependency on MYB expression and that such variability is dictated by the nature of the driver mutation. However, whether this difference in MYB dependency is a general trend in AML remains to be further elucidated. Here, we investigate the role of MYB in human leukaemia by performing siRNA-mediated knock-down in cell line models of AML with different driver lesions. We show that the characteristic reduction in proliferation and the concomitant induction of myeloid differentiation that is observed in MLL-rearranged and t(8;21) leukaemias upon MYB suppression is not seen in AML cells with a complex karyotype. Transcriptome analyses revealed that MYB ablation produces consensual increase of MAFB expression in MYB-dependent cells and, interestingly, the ectopic expression of MAFB could phenocopy the effect of MYB suppression. Accordingly, in silico stratification analyses of molecular data from AML patients revealed a reciprocal relationship between MYB and MAFB expression, highlighting a novel biological interconnection between these two factors in AML and supporting new rationales of MAFB targeting in MLL-rearranged leukaemias.

Clinical Characteristics and Outcomes of Myeloid Neoplasms with Mecom Rearrangement: Results from a Nationwide Multicenter Study

MECOM rearrangements are detected in myeloid neoplasms [myelodysplastic neoplasm (MDS) and acute myeloid leukemia (AML)] which are associated with dismal prognosis. Classic MECOM rearrangements include inv(3)(q21q26.2) and t(3;3)(q21;q26) while non-classic subtypes are 3q26.2/ MECOM rearrangement with other partners. Both classic and non-classic rearrangements result in MECOM overexpression which involves in the process of leukemogenesis. Recently, the World Health Organization classification 2022 categorized myeloid neoplasms with these genetic abnormalities as “AML with MECOM rearrangement” regardless of blast count. We aim to explore frequency, clinical characteristics and outcomes including treatment response in this AML subtype among Thai myeloid neoplasms. AML data was collected from a national registry which were conducted by Thai acute leukemia working group. Other than AML with MECOM rearrangements, AML was categorized as favorable, intermediate, and adverse risk groups according to European Leukemia Network 2022. MDS data was collected from a multicenter study group involving 4 medical centers. Other than MDS with MECOM rearrangements, MDS was categorized into 5 risk groups according to R-IPSS score system (very low, low, intermediate, high and very high-risk groups). Myeloid neoplasms with MECOM rearrangements were analyzed among their diseases and grouped together and compared to MDS and AML cohorts. A total of 9 cases of myeloid neoplasms with MECOM rearrangement were detected. Among non-M3 AML cases, there were 6 cases with MECOM rearrangement from 746 non-M3 AML (0.8%) while 3 cases were detected in 163 MDS (1.8%). Seven of 9 cases (78%) were female gender. Five of 9 cases were classic MECOM rearrangement [1 case with inv(3)(q21q26.2) and 4 cases with t(3;3)(q21;q26)] while the other 4 cases were non-classic rearrangements [3 cases with t(3;21)(q26.2;q22) and 1 case with t(3;7)(q26;q21)]. In the AML cohort, AML with MECOM rearrangement showed lower white blood cell, but higher platelet counts compared to other groups (favorable, intermediate, and adverse risk groups) ( Table 1). Among AML cases receiving intensive chemotherapy, MECOM rearrangement subgroup showed lower complete response (CR) rate compared to others favorable, intermediate, and adverse risk groups. (0% vs. 77.3% vs. 37.6% vs. 23.8%; p<0.001). Of note, among 6 AML with MECOM rearrangement, there were 4 patients who received intensive chemotherapy but none of them responded to the treatment. In the MDS cohort, MDS with MECOM rearrangement showed lower hemoglobin and platelet counts compared to other groups ( Table 2). Among 3 MDS with MECOM rearrangement, one patient received azacitidine with investigational drug (sabatolimab/placebo) and achieved complete hematologic response. He eventually progressed after 12 cycles of the treatments and subsequently died. When combining 3 MDS and 6 AML with MECOM rearrangement as one group and compared survival rate with others: survival rate of 9 myeloid neoplasms with MECOM rearrangementis worse than the adverse group of AML and the very high risk group MDS with a 1-year survival rate of 22% ( Figure 1 and 2). In conclusion, myeloid neoplasms with MECOM rearrangements are rare with the frequency of 0.8% in non-M3 AML and 1.8% in MDS. This subtype is more common in female gender. The prognosis of myeloid neoplasms with MECOM rearrangement is dismal with a 1-year survival rate of 16.7% in AML and 6-month survival rate of 33% in MDS. Chemotherapy should be avoided in this subtype due to non-responsiveness, hypomethylating agent showed benefit and can be considered as a bridging treatment before stem cell transplantation. Novel therapy targeting MECOM gene should be further explored to improve outcomes in this AML subtype.

Unbiased Subtyping of AML: Unraveling Genomic and Transcriptomic Features for Precision Medicine and Targeted Therapies Using Beat-AML and TCGA Data

Background: Acute Myeloid Leukemia (AML) is a heterogeneous hematological malignancy with variable clinical outcomes. Identifying patient subsets with relevant prognostic outcomes and understanding their molecular characteristics is crucial for developing targeted therapies. This study aimed to characterize molecular subtypes in AML patients using unsupervised clustering analysis, and investigate the relationship between transcriptomic features, signaling/immune pathways, recurrent DNA variants, and prognostic potential. Here, we show the potential of applying an integrative multi-omics approach to define the disease biology of a unique poor prognostic subtype of AML to uncover potential targeted therapeutic strategies. Methods: An unsupervised dimensionality reducing approach (non-negative matrix factorization) followed by consensus clustering was performed on the Beat-AML (discovery dataset, N=462) and TCGA-AML (validation dataset, N=173) RNAseq data from patient samples to identify transcriptomic-based molecular subtypes. The top 1000 most variable genes were used to reduce noise. The biological significance of gene expression in the identified subtypes was analyzed using GSVA on Hallmark, KEGG and Reactome databases. Immune cell type abundance scores were calculated by xCell method. The presence of recurrent DNA variants was evaluated in each subtype. The association between subtypes and clinical outcome was tested using Kaplan Meier method and a log-rank test. The findings were then reproduced in the TCGA AML cohort. Results: Analysis in the Beat-AML cohort revealed 5 clusters, including cluster 3 (n=86; comprising 20% of the AML cases) which showed worse overall survival (OS) compared to the other subtypes (HR (95% CI) = 1.41 (1.03, 1.92, P=0.03). A co-mutation in the genes FLT3, NPM1 and DNMT3A was enriched in this subtype. Gene sets analysis showed enrichment of several pathways key to the mechanism of action of therapeutic antibodies, including enhanced enrichment of the complement pathway, NK cell-mediated cytotoxicity, and FCGR-mediated phagocytosis in this subgroup. Additionally, activation of oncogenic signaling (PI3K/AKT/MTOR, TGFβ, NOTCH), and metabolic pathways (fatty acid and oxidative phosphorylation) were enriched in this subgroup. Immune cell signatures showed elevated expression levels of M2 macrophages. The results were successfully reproduced in the TCGA AML cohort, validating the findings. Conclusion: This study identifies a distinct poor prognostic patient subpopulation in AML characterized by activation of immune and cancer related pathways, and enrichment of a specific co-mutation genotype. While co-mutation of NPM1/FLT3-ITD has been identified as an intermediate-risk genotype and DNMT3A mutation alone is associated with poor prognosis (Ley et al., 2010, N Engl J Med), the triple co-mutation enriched in cluster 3 of our analysis may itself lead to poor prognosis (Loghavi et al., 2014, J Hematol Oncol) but the biological features of this subtype have yet to be described. The transcriptome data revealed patients in cluster 3, which may include nearly 20% of AML, exhibited activation of oncogenic signaling pathways, metabolic stress, and an immunosuppressed tumor microenvironment - all characteristics of aggressive disease; there is also enrichment of genes driving NK cell-mediated cytotoxicity, FCGR-mediated phagocytosis, and complement cascade, highlighting a potential opportunity for antibody-based immune effector function-dependent interventions. Replication of the results in an independent cohort strengthens the robustness of the findings. This study enhances our understanding of AML heterogeneity and may have implications for developing targeted antibody-based strategies to treat high-risk subtypes, such as this specific molecular subgroup, ultimately improving patient outcomes in AML.

Acute Myeloid Leukemia with Inv(3) or t(3;3): A Clinical and Cytogenetic Characterization of 40 Patients

Background: Acute myeloid leukemia (AML) with inv(3) or t(3;3) is an extremely aggressive and rare subtype of AML. Leukemogenesis in inv(3) AML is in part due to juxtaposition of the GATA2 enhancer in close proximity to the MECOM/EVI1 promoter; as the result, the fusion drives EVI1 overexpression and uncontrolled proliferation of myeloid progenitors. Given its rarity, published clinical outcome data are limited. To improve our understanding of the clinical outcomes of inv(3) AML, we conducted a retrospective study to describe the clinical features of a cohort of newly diagnosed inv(3)/t(3;3) AML patients treated at Stanford Cancer Center between 2002 and 2023. Methods: Data were retrospectively collected from databases associated with the Stanford Health Care electronic health record (EHR), and diagnosis dates came from the Stanford Cancer Registry. Inv(3)/t(3;3) AML diagnosis was defined according to WHO criteria as the presence of ≥20% blasts in the bone marrow (BM) or peripheral blood (PB) and the presence of inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2) on cytogenetic analysis at initial diagnosis. Response to first induction, salvage, and consolidation therapy as well as allogeneic hematopoietic stem cell transplant (alloHSCT) were captured. CR was defined as the presence of <5% of blasts in the BM evaluation by morphological evaluation after each line of treatment. Overall survival (OS) was defined as time from diagnosis to death from any cause. A descriptive study of the main demographic, clinical, hematologic, and cytogenetic variables of patients who received intensive and non-intensive therapy was performed. The analysis was performed using R 4.3.1. A p value of < 0.05 was defined to be statistically significant. Results: A total of 40 patients met inclusion criteria including 68% (n=27) with de novo AML, 25% (n=10) with secondary AML from an antecedent hematological disorder and 7.5% (n=3) therapy-related AML. Clinical characteristics of the patients are summarized in Figure 1A. The median age at time of diagnosis was 56 (range, 19-78). There was a slight female predominance, with 58% (n=23) female and 42% (n=17) males. Ninety-three percent of patients had an ECOG score ≤1 (n=37). Median leukocyte, hemoglobin, and platelet counts were 4.5 K/uL, [1.3-761], 9.4 g/dL, [3.4-13.6], and 98 K/uL [9-592], respectively. The median blast percentage in BM and PB was 49% [13-98] and 30% [0-95], respectively. Inv(3)/t(3;3) was the only cytogenetic aberration in 10 patients (25%) and chromosome (chr) 7 abnormalities were the most common additional aberration, present in 22 patients (55%). CR after the first induction therapy was achieved in 9 patients (22%); 6 patients (15%) died during or shortly after induction therapy, and the remaining 25 (63%) were resistant. Salvage therapies for patients included re-induction with 7+3, FLAG-Ida, mitoxantrone-containing regimens, clofarabine-based protocols, or combination of hypomethylating agents with venetoclax. Seventeen (43%) patients underwent alloHSCT (10 in CR and 7 with active disease). When comparing intensive vs non-intensive induction regimen, no statistical significance was identified for complete remission (CR) rate (p= 1.00), overall survival (OS) rate (p=0.65) or relapse-free survival (RFS) rate (p=0.86). Similarly, no statistically significant prognostic factors (age, WBC, BM blast %, platelet count, hemoglobin, ANC, or time to transplant) for OS were identified. Interestingly, chr 7 abnormalities in inv(3) patients conferred a worse prognosis in this cohort (OS 41% vs. 59%, p= 0.0064, Figure 1B). This association was confirmed using a multiple Cox proportional hazards regression adjusting for age and blast percentage. Moreover, while chr 7 abnormality was also associated with a lower response rate (CR 14% vs. 40%), it did not reach statistical significance (p=0.12). Conclusions: In summary, de novo AML inv(3)/t(3;3) is a rare and aggressive subtype of AML conferring extremely poor prognosis. There appears to be an increase of incidence in females affecting a slightly younger demographic. Consistent with previous studies, the presence of chr 7 abnormalities was associated with even worse outcomes, which interestingly also drive EVI1 overexpression. The exact link between 3q26 loci rearrangements and chr 7 abnormalities warrants further investigation.

Kme-0584, a Highly Potent IRAK1/IRAK4/panFLT3 Inhibitor, Is a Promising Clinical Candidate for Hypomethylating Agent Plus Venetoclax Resistant AML/MDS Patients

Leukemic cells exhibit dysregulation of innate immune signaling pathways upon diagnosis, and these pathways become further activated in drug resistance. The Interleukin-1 receptor associated kinase 1 and 4 (IRAK1/IRAK4) kinase complex is part of a critical signaling node that becomes activated in these dysregulated pathways (reviewed in J Bennett and DT Starczynowski, Curr Opin Hematol 2022). IRAK4 inhibitors are currently under investigation for the treatment of Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) and have shown encouraging, though modest responses in clinical studies. We have recently demonstrated that limited responses to IRAK4 inhibitors in the leukemic setting can be explained by a compensated upregulation and activation of IRAK1, and the need to inhibit both IRAK1 and IRAK4 to achieve maximal therapeutic efficacy (JR Bennett et. al., Blood 2023 https://doi.org/10.1182/blood.2022018718). KME-0584 is a highly potent IRAK1/IRAK4/panFLT3 inhibitor that exhibits superior potency and therapeutic efficacy vs. IRAK4 inhibitor compounds in the FLT3 WT as well as the FLT3 mutant setting. KME-0584 exhibits >100-fold selectivity vs. 89% of the Kinome as measured in the Reaction Biology 374 kinase panel, with an IC50 of 23, <1.29, and <0.5nM at IRAK1, IRAK4, and FLT3 respectively. Higher kinase selectivity is seen in cell-based assays vs. biochemical assays, where high potency kinase antagonist activity is also observed against PHKg1, PDGFRβ, RET, CLK1, and CLK4. While IRAK4-selective compounds fully antagonize NF-κB signaling through the TLR pathway, KME-0584 completely inhibits NF-κB signaling through both the TLR and IL1-receptor pathways, indicating complete inhibition of multiple receptor pathways converging on NF-κB requires both IRAK1 and IRAK4 antagonism. In primary patient cell lines from FLT3 wildtype (WT) patients, KME-0584 inhibits leukemia stem cell progenitor function as measured by the colony formation assay in methylcellulose with higher potency than IRAK4 inhibitor compounds that lack IRAK1 activity such as CA-4948 (Emavusertib). This activity is independent of patient mutational status. Importantly, high potency in primary patient cell lines is observed independent of the presence of U2AF1 or SF3B1 spliceosome mutations known to drive expression of the activated IRAK4 Long isoform of IRAK4 (Fig 1), implying that the IRAK1/4/panFLT3 inhibitor KME-0584 will have activity across a broader patient population than an IRAK4 inhibitor. We have previously described an IRAK1/4 gene signature that is highly expressed in patients with myelomonocytic subtypes (M4 FAB) of adult and pediatric AML and with an antecedent MDS (JR Bennett et. al., Blood 2023 https://doi.org/10.1182/blood.2022018718). The IRAK1/4 gene signature is reversed after treatment of THP1 cells for 24 hours with KME-0584. Given that monocytic-like subtypes of AML are resistant to Venetoclax plus Azacitidine (VEN/AZA) (S Pei et. al. Cancer Discovery, 2020), these data suggest that KME-0584 has the potential to be effective across a broader range of relapsed/refractory AML/MDS patients than previously examined IRAK4 inhibitors. KME-0584 exhibits superior potency and efficacy to gilteritinib in the FLT3-ITD (D835Y) xenograft model after QD oral dosing, with sufficient PK and oral bioavailability across multiple species to support QD or BID dosing in the clinic. KME-0584 does not inhibit any of the major or minor cytochrome P450 enzymes at anticipated clinical concentrations and early indication from ongoing GLP toxicology studies suggest that it could be safely administered in humans. A clinical study of KME-0584 in relapsed/refractory AML and HR-MDS is currently planned to start in 1H 2024.

Long-Term Outcome of 1296 Patients with Newly Diagnosed with APL: A Harmony Alliance Study

Background: APL, a rare and aggressive subtype of acute myeloid leukemia (AML), is now curable in 75-90% of patients using targeted agents [All-trans retinoic acid (ATRA)/Arsenic Trioxide (ATO) or ATRA combined with chemotherapy (ATRA+Idarubicin, AIDA-based)]. Despite significant advances in survival, most reports are based on low patient numbers, which limit reliability of data. Aims: to study factors associated with outcome in a large patient cohort diagnosed with APL, and treated according to two European trials (UK AML-17 and GIMEMA APL0406), or recorded in national registries from 6 countries, enrolled in the HARMONY database. Methods: The Harmony platform includes to-date 1868 patients with APL, diagnosed between 2007 and 2020. The present analysis was performed on 1296 patients, who met the data quality requirements, by evaluating the list of variables shown in Table 1. Patients were treated according to APL0406 and AML17 clinical trials, or were included in the Study Alliance Leukemia (SAL), Swedish Cooperative Group or AML study Group (AML-SG) registries. After acquisition from the sources, data were harmonized and transformed using an Observational Medical Outcomes Partnership Common Data Model, and registered in the HARMONY Big Data Platform. Results: Of 1296 patients, 562 were treated with ATRA-ATO (median age 51 yrs, range 16-94; M:F ratio 1), and 605 with ATRA-Idarubicin (AIDA, median age 50 yrs, range 17-86; M:F ratio 1) (p for age groups according to treatment=0.3538). According to Sanz risk-score, 250 patients (44.4%) were low-risk (LR), 258 (45.9%) intermediate-risk (IR), and 50 (8.9%) high-risk (HR) in the ATRA-ATO cohort. The AIDA cohort included 191 LR (31.5%), 235 IR (38.8%), and 171 HR (28.3%) patients (p for risk groups according to treatment: < 0.001). Treatment data were not available in 8 patients (1.3%) The 10-year overall survival (OS) was 90% and 77% in ATRA-ATO vs AIDA groups, respectively (p<0.001, figure 1), while event-free survival (EFS) was 86% and 67%, respectively (p<0.001). At a median follow-up of 4.5 yrs (range 0.02 - 10.2), OS and EFS in patients treated with ATRA-ATO was similar in the three Sanz-risk classes (OS: LR: 94.6%, IR: 91%, HR: 85.6%, p=0.233, EFS: LR: 93.1%, IR: 88.7%, HR: 85.7%, p=0.319). The median follow-up of patients treated with AIDA was 6 yrs (range: 0-14.2). OS was 87.8% in LR, 83.5% in IR, and 74.8% in HR (p=0.004), while EFS was 72% in LR, 74.8% in IR, and 67.2% in HR (p=0.102). OS was significantly associated with age (<50 yrs: n=611, 50-69: n=516, >70: n=169), both overall (p<0.001), and within treatment groups (ATRA-ATO: p<0.001, AIDA: p<0.001). EFS also correlated with age subgroups, both overall (p<0.001) and according to treatment (ATRA-ATO: p<0.001, AIDA: p<0.001). No differences in OS were detected in between de-novo and secondary APL (p=0.746) both in ATRA-ATO (p=0.720) and in AIDA (p=0.766) cohorts. Patients treated outside the clinical trial context had inferior outcomes when compared to clinical trials, with a significant higher rate of early deaths (ED, 10% vs 2.8%, p<0.001), and inferior survival with major differences for the AIDA cohort (8-year OS: 73.6% vs 88.2%, p<0.001, EFS: 69.3% vs 71.7%, p=0.291), versus the ATRA-ATO cohort (8-year OS: 88% vs 93.3%, p=0.010, 8-years EFS: 80.6% vs 92.4%, p<0.001). The multivariate analysis for OS showed that age (50-69 vs <50 years, p<0.001, HR 4.2; >70 vs <50 years p<0.001, HR 8.9), Sanz-risk score (High vs Low/Intermediate p<0.001, HR 2), treatment type (AIDA vs ATRA-ATO p=0.001, HR 1.9) and treatment context (clinical trial vs non-clinical trial p=0.005, HR 1.7) were independent predictors of OS. The multivariate analysis for EFS showed an independent correlation with type of treatment (AIDA vs ATRA-ATO, p<0.001 HR 2.6), age (50-69 vs <50 years, p<0.001, HR 2.1; >70 vs <50 years p<0.001, HR 4.8) and Sanz-risk score (High vs Low/Intermediate p=0.005, HR 1.5), but not with treatment context. In multivariate analysis treatment type was the only predictive factor for early deaths (AIDA vs ATRA-ATO, p=0.047 HR 2.04). Summary/Conclusions: The analysis at long-term of the Harmony APL registry confirmed that patients treated with the ATRA-ATO chemo-free regimen present a significant survival advantage vs the AIDA regimen, with reduced ED rates and prolonged OS, independent of Sanz-risk score. Patients enrolled in clinical trials presented improved survival both in AIDA and in ATRA-ATO cohorts.

Machine Learning-Facilitated Cross-Panel Automatic Classification at Both Sample and Cell Level in Acute Myeloid Leukemia

Introduction Flow cytometry (FC) immunophenotyping is essential for accurate and prompt diagnosis of acute myeloid leukemia (AML), with up to 95% of AML patients showing detectable aberrant immunophenotype (IP) by FC. Current manual and subjective analysis of clinical FC data, however, lacks reproducibility and leads to interpretation variation. In previous studies, we showed that supervised machine learning (ML) approaches can detect IP abnormalities and identify the acute leukemia subclassification accurately at the specimen level. We also found that comparable classification performance can be achieved using only a subset of markers of the reagent panel (Ko et al., 2018; Monaghan et al., 2022). To interpret the ML classification results, we have developed data clustering and discriminative learning methods to identify and visualize the diagnostic cell populations in a 2D space for hematopathology review (Ji et al., 2020; Lee et al., 2018). Built upon these approaches, this study reports our findings on developing an interpretable cross-panel ML classification model to support timely and accurate diagnosis and subclassification of AML. Materials and Method The AML diagnosis model at the sample level utilized FC data obtained from 53 bone marrow (BM) aspirate samples from Roswell Park Comprehensive Cancer Center (RPCCC) and 50 BM aspirate samples from UPMC. Data from RPCCC were acquired using a Beckman Coulter Navios EX and measured with the ClearLLab10C panel; while data from UPMC were acquired using a BD FACSCantoII and measured with a UPMC-developed diagnostic panel. We used only the common parameters collected within these 103 FC data sets and applied a previously published Gaussian Mixture Model-Support Vector Machine (GMM-SVM) based approach to train a cross panel sample-level classification model for AML diagnosis (Ko et al., 2018; Monaghan et al., 2022). Three-fold cross-validation was conducted to assess the model performance, using sensitivity, specificity, classification accuracy, and area under the receiver operating characteristic curve (AUC). For the cell-level visualization and analysis, all samples in the training set were pooled together, before a uniform manifold approximation and projection (UMAP) process was applied to transform and visualize the cell populations within the samples. Specifically, 26 non-neoplastic and 27 AML samples from the M2 panel of the ClearLLab10C from the RPCCC cohort were combined for the UMAP visualization. Then the FlowSOM unsupervised clustering method and the DAFI semi-supervised clustering method (Lee et al., 2018) were applied to identify all of the cell populations that have significant differences between the AML samples and the non-neoplastic controls. Results The cross-panel AML versus non-neoplastic classifier, combining FC data from UPMC and RPCCC achieved exceptional performance. The AML detection model using all parameters on the ClearLLab10C panel achieved an AUC of 100%. When using only the overlapping parameters between the ClearLLab10C and the UPMC panels, the classification model achieved an AUC of 99.5%, while the cross-panel classifier achieved an AUC of 99.1% (Table 1). Specimen-level visualization confirmed the separation of AML versus non-neoplastic controls. The UMAP plots, created by pooling non-neoplastic and AML samples, exhibited distinct patterns for different AML subtypes (M1 to M5) and a genetic mutation (e.g., AML IDH1), which were distinct from control samples. Using the automated gating analysis, cell populations identified as different between AML and control samples were characterized and visualized in 2D-by-2D scatter plots for straightforward interpretation and assessment based on their immunophenotype. Conclusion Our study demonstrated the feasibility to develop and assess a ML classification model for AML detection and diagnosis using overlapping markers in FC data across reagent panels and sites. Advanced visualization and automated gating analysis enabled the interpretation of the ML classification by identifying and characterizing the diagnostic cell populations and their phenotypic heterogeneity, providing insights into AML heterogeneity at both the specimen and the single cell level. These findings highlight the potential of ML capability for improving patient care by timely and accurate diagnosis and subclassification of AML.

Feasibility, Safety and Predictors of Outcomes of Patients with Newly Diagnosed Acute Myeloid Leukemia Discharged “Early” after Intensive Induction Therapy

Background: Patients (pts) treated with intensive induction therapy for newly diagnosed acute myeloid leukemia (AML) traditionally remain admitted until an absolute neutrophil count (ANC) >0.5 x 10 3/L. Although a small number of centers reported on pt discharge with ANC <0.5 x 10 3/L via an “early” discharge (DC) program (EDP) post-intensive induction, little is known about the safety and outcomes among this group of pts, including the rates and predictors of post-DC complications (e.g., febrile neutropenia (FN), microbiologically proven infection) and both early- and long-term outcomes. We sought to describe our experience with an EDP and identify predictors of differential outcomes. Methods: We conducted a single-center, retrospective study of pts with newly-diagnosed AML admitted to Yale Cancer Center (YCC) for intensive induction during December 2014-January 2023. Re-induced patients were excluded. Patients were eligible for the EDP if they: 1) were afebrile x 7 days, 2) required no parenteral therapies, 3) required transfusion support less than daily, 4), lived within 60 minutes of YCC with “average” traffic, and 5) had a dedicated care giver available 24 hours per day with reliable transportation to/from clinic. Chi-square and Wilcoxon rank-sum tests were applied to categorical and continuous variables, respectively. Overall survival (OS) was estimated using the Kaplan-Meier method and was compared using log-rank test. Statistical significance was determined by p-value <0.05. Results: A total of 188 pts were evaluated. Amongst this population, 99 (52.7%) pts were DC under the EDP, while 89 (47.3%) were not. To remove confounding from peri-DC ANC kinetics, we focused on pts DC with an ANC <0.1 x 10 3/L (n=53, “EDP pts”) and compared them with non-EDP pts (DC ANC >0.5 x 10 3/L). There were no significant differences in age, sex, ECOG performance status (PS), baseline LVEF on echocardiography or other comorbidities. With regards to disease-specific factors, there was no difference in pre-induction blood counts (e.g., WBC 7.2 vs. 8.1, p=0.610), serum creatinine, uric acid, lactate dehydrogenase, AML subtype, or disease biology e.g., karyotypic or molecular features (Table 1). Pts received 7+3 without (47%) or with (27%) other therapy, while 22% received liposomal daunorubicin/cytarabine. There were no differences in the rates of regimens used between EDP and non-EDP pts. The incidence of induction-related complications like FN (81.1% vs 89.9%, p=0.220), DIC, TLS, and bleeding were similar between groups, although there was a trend toward more documented infection among non-EDP pts (48.3% vs 30.2%, p=0.052). Findings on the D14 marrow (performed in 79% of pts) were similar between groups (chemoablation, p=0.131; D14 blast %, p=0.546). Median days admitted from induction to DC was nearly a week less for EDP pts (22 [interquartile range (IQR): 19-29] vs 28 days [IQR: 25-32], p<0.0001). Amongst EDP patients, 12 (22.6%) were re-admitted, 10 (83%) due to FN with 4 of these pts having an infectious source identified. The median duration of re-admission was 6.5 days (IQR: 4.5-13). When considering re-admissions prior to ANC/count recovery or initiation of consolidation, EDP pts still had lower time spent in hospital prior to eventual count recovery (24 vs 28, p<0.0001). ANC at DC did not predict OS amongst the larger cohort (n=188, p=0.949), but was associated with re-admission amongst pts DC with ANC <0.5 in univariate analysis ( p=0.02). Time to DC and ANC at DC were strongly associated amongst the larger cohort (n=188, p=0.01 from linear regression) and pts DC with ANC <0.5 ( p<0.00001 from linear regression)(Figure 1). Although non-EDP pts had a shorter time to first response (33 vs 36 days, p=0.020), there were no differences in rates of CR/CRi/CRh (73.0% vs 60.4%, p=0.167) when compared with EDP pts. Similarly, there were no differences in early (60- and 90-day) mortality, or median OS between EDP and non-EDP pts (32.3 vs 26.4 months, p=0.695)(Table and Figure 1). Conclusions: Our study shows that pts treated intensively for newly-diagnosed AML and meeting several criteria can be safely DC prior to count recovery without detriment to early mortality or OS. EDP pts spent up to a week more out of hospital when compared with non-EDP counterparts. Healthcare utilization, cost effectiveness and patient satisfaction evaluations of the EDP are underway.

Correlation Revealed between Gemtuzumab-Ozogamicin Efficacy and CD33+ Expression in AML Primary Samples through a Novel AML in Vitro Model

Acute myeloid leukemia (AML) is one of the most difficult blood cancers to treat with increasing incidence in recent years. The disease is characterized by a clonal hematopoietic aggressive disorder of progenitor and stem cells. The prognosis for patients with AML is generally poor. After the first remission, most patients will develop recurrent disease, often within the first year 1. Recent molecular deconvolution of the mechanisms underlying AML disease have impacted the way AML patients are managed in the clinic, going from chemotherapy to the implementation of monoclonal antibodies and antibody-drug conjugates (ADC) 2. One of the limitations to ADC development for AML patients is the lack of AML-specific antigens. CD33 is a recent target which, although being expressed by other normal hematopoietic cells, is also strongly expressed in 80-90% of all AML cells. For these reasons gemtuzumab-ozogamicin (Mylotarg), a monoclonal antibody targeting CD33 covalently linked to the toxin calicheamicin, is the only FDA approved ADC for AML treatment 2. Although these treatments have had an impact on patient care, new therapeutic strategies are required to succeed in effectively treating AML. To date, several AML models have been exploited to predict patients' response 3. The model portfolio comprises in vivo and ex vivo models; however, the difficulties in culturing AML primary cells while maintaining clonal heterogeneity often limit the ability of the assay to recapitulate the disease. The development of a functional experimental model capable of mimicking the heterogeneity and complexity of AML with translational potential is still a major challenge. To overcome the lack of clinically relevant translational models that can stratify sensitive and resistant tumors across different treatments, we developed our proprietary assay, Champions AML VitroScreen ®, to test therapeutic candidates in primary AML samples 4. Our bank of primary, deeply characterized AML samples recapitulates the diversity of multiple AML subtypes from a phenotypic and molecular standpoint. This assay allows for evaluation of cell viability, proliferation, and clonal composition in response to therapeutic intervention using a variety of validated endpoint analyses. To challenge the robustness of the assay, we used the VitroScreen platform to test Mylotarg, in primary AML samples used as models for the different AML subtypes including M1, M2, M3, M4 and M5. To select appropriate AML models, we evaluated the expression of CD33 in AML blasts by high complexity flow cytometry. CD33+ high, intermediate, and low expressing models were included in the study. We measured the IC50 for Mylotarg in more than 20 AML models and correlated the marker expression with drug efficacy. Dose-dependent responses to Mylotarg were observed across multiple models by evaluating cell growth and viability using the CellTiter-Glo ® assay. As additional endpoints cell proliferation, apoptosis, and phenotyping by flow cytometry were included to better interpret Mylotarg mechanism of action. Based on the assay results, primary AML models were classified into different categories, respectively responder, low responder and resistant, based on the IC50 value, and a strong positive correlation between sensitivity to Mylotarg and CD33+ expression was found. The use of deeply characterized AML patient primary samples within the AML VitroScreen ® platform allowed for high throughput drug testing, which represents the ideal clinical translational setting in preclinical research. The use of this innovative system for patient selection and drug screening will support the development of new and tailored therapeutic strategies for patients with primary or recurrent AML, leading to improved patient outcomes.

Dissecting Subtype-Specific Tumor-Time Interactions and Underlying Hidden Drivers in Pediatric Acute Myeloid Leukemia Via Single-Cell Multi-Omics

Introduction: Pediatric acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by various chromosomal abnormalities and somatic mutations. Investigating the origin of tumorigenesis and deciphering the complex interplay between AML cells and their surrounding tumor immune microenvironment (TIME) is crucial to understand the underlying mechanisms driving disease progression and response to therapy. Recent advancements in single-cell multi-omics technologies enabled us to characterize the transcriptional (GEX) and epigenetic (ATAC) landscapes of both tumors and TIMEs. Leveraging our in-house algorithm, scMINER (single-cell Mutual Information-based Network Engineering Ranger, Ding et al., 2023), this study aims to build a comprehensive cell type atlas in diverse pediatric AML bone marrow samples and construct intra- and inter-cellular networks, enabling the identification of hidden drivers that shape subtype-specific AML-TIME interactions. Methods: Using CD45 and CD33 as selective markers by flow cytometry, we have successfully isolated blasts and enriched for T cell and B cell populations for 10X single-cell multiome profiling across AML molecular subtypes, including UBTF-TD (n=1), NPM1 (n=1), and KMT2A rearrangement (n=1). We interrogated the GEX data using scMINER, including two key steps: (i) mutual information-based clustering analysis (MICA) and (ii) mutual information-based network inference engine (MINIE). MICA characterizes the intrinsic nonlinear similarity of gene expression distributions among cells, enabling a high resolution of clusters. MINIE reverse-engineers cluster-specific intracellular gene networks and infers protein activity of transcription factors (TF) or signaling factors (SIG) drivers based on the expression of its predicted regulon targets in the corresponding cluster. The activity profiles overcome the dropout effects of single-cell RNA-seq data and reflect protein activities across all cells. ArchR is used to analyze ATAC data. CellChat is used to construct and evaluate intercellular networks. Results: AML cells from three samples were divided into four major clusters, and we labeled them as NPM1, KMT2A, UBTF_one, and UBTF_two based on sample subtypes (Fig.1A). MICA managed to obtain distinct AML cell sub-clusters. Comparison with single-cell profiles of normal hematopoietic cells and annotation with marker genes revealed their correspondence with different myeloid developmental stages. Notably, the KMT2A cluster shared a high similarity in cell type proportion with the UBTF_two cluster, and the NPM1 cluster is closer to the UBTF_one cluster. This result demonstrated significant tumor heterogeneity within and across AML molecular subtypes. With the help of intracellular gene networks generated by MINIE, we identified multiple subtype-specific hidden drivers by differential activity analysis. For example, KMT2A and UBTF_two clusters exhibited elevated SAMHD1 levels, consistent with previous GSE data (Zhang et al., 2022). Chromatin accessibility analysis of ATAC data showed increased peaks near SAMHD1 in KMT2A and UBTF_two clusters, further supporting this observation. IRF8 showed a similar expression pattern to SAMHD1, suggesting its context-dependent role across AML subtypes. Accurate annotation of myeloid and immune cells greatly enriches the preciseness of the AML and TIME interaction patterns, which vary across AML molecular subtypes (Fig.1B). Interestingly, cells originated from KMT2A cluster exhibited similar interaction patterns to UBTF_two, while NPM1 and UBTF_one were grouped. This result indicated that different AML molecular subtypes may partially utilize the same mechanisms of tumor-TIME interaction, resulting in similar tumor phenotypes. Conclusions: Accurate clustering and well-defined annotation of myeloid and immune cells enabled us to build a comprehensive cell type atlas in AML samples. The subtype-specific network and the following analysis identified candidate hidden drivers that possibly contribute to the heterogeneity of tumor populations. AML subtypes sharing the same hidden drivers have similar cell type proportions and interaction patterns with TIMEs. This observation implies that AML subtypes share cell type-specific dependencies, which can be therapeutic targets to overcome the refractoriness posed by tumor heterogeneity.

Genetic Determinants of Dependency on Regulated Protein Synthesis in Leukemia Stem Cells

Acute myeloid leukemia (AML) is initiated and sustained by rare self-renewing leukemia stem cells (LSCs). LSCs do not resemble HSCs, but rather more mature hematopoietic progenitor cells that usually have no self-renewal potential. However, during leukemogenesis, LSCs become aberrantly self-renewing, but the mechanisms that allow for this are poorly understood. We recently used models of AML driven by AML-ETO9a and MLL-AF9 to show that LSCs may co-opt from HSCs a dependence on regulation of protein synthesis mediated by CD99 (ASH 2021, #2222), which we identified as a therapeutic target highly expressed in LSCs (PMID: 28123069). However, it remains unclear if LSCs from specific subtypes of AML are particularly dependent on regulated protein synthesis. In an analysis of transcriptome data from the ECOG 1900 cohort, we found that certain mutations ( KIT, PTEN, AML-ETO, IDH1/2 and FLT3-ITD) were associated with the highest levels of CD99, raising the possibility that such AML subtypes are more dependent on CD99. In the TCGA AML cohort, CD99 expression was significantly higher in AML patients with FLT3-ITD mutations but not NRAS mutations ( Fig. 1A). To test if LSCs from AMLs driven by mutations in FLT3 or NRAS differ in their dependence on regulation of protein synthesis by CD99, we crossed Cd99 -/-(CD99 KO) mice with two mouse models of AML driven by these alleles to generate Cd99 -/-; Mx1-Cre; Tet2 fl/fl; Flt3 ITD/+and Cd99 -/-; Mx1-Cre; Tet2 fl/fl; Nras LSL-G12D/+ mice (CD99 KO TET2/FLT3 and CD99 KO TET2/NRAS). TET2 was chosen as a cooperating allele given its neutral association with CD99 in the above human datasets, and it additionally co-occurs frequently with mutations in FLT3 or NRAS. To generate AML in both models, Tet2 was deleted with polyinosine-polycytidylic acid injections, followed by transplant of 4 million bone marrow (BM) cells into irradiated wild-type (WT) mice. At 16 weeks, recipients of CD99 WT TET2/FLT3 BM developed an expansion of aberrant donor-derived Mac-1 + c-kit + cells in the peripheral blood (PB), and BM aspirates revealed an accumulation of LSK CD48 +CD150 - cells that harbor LSC activity in this model ( Fig. 1B). In contrast, recipients of CD99 KO TET2/FLT3 BM exhibited significant depletion of these aberrant populations (P=0.0055 and P=0.019 respectively, n=10 for each group). Recipients of CD99 WT TET2/FLT3 BM also exhibited increased leukocytosis and thrombocytopenia, which were attenuated in recipients of CD99 KO TET2/FLT3 BM (P=0.046 and P=0.042, respectively). In contrast, there was no difference between recipients of CD99 WT TET2/NRAS and CD99 KO TET2/NRAS BM in emergence of the aberrant Mac-1 + c-kit + cells and thrombocytopenia (P=0.3237 and P=0.9752, respectively, n=10 for each group). These data suggest that loss of CD99 may impair leukemogenesis in TET2/FLT3 but not TET2/NRAS AMLs. To determine if CD99 is required for LSC function, we performed secondary transplants of one million BM cells from primary recipients into lethally irradiated WT recipients. Mice transplanted with CD99 KO TET2/FLT3 BM, compared with those transplanted with CD99 WT TET2/FLT3 BM, demonstrated fewer donor-derived cells in the PB. While CD99 WT TET2/FLT3-transplanted mice succumbed to AML at a median of 11 weeks, all CD99 KO TET2/FLT3-transplanted mice survived beyond 16 weeks. PB smears from CD99 KO TET2/FLT3 mice demonstrated leukocytes with condensed chromatin and lobulated nuclei, indicative of restored hematopoietic maturation. Finally, in vivo O-propargyl-puromycin assays showed that loss of CD99 resulted in increased protein synthesis in LSCs (LSK CD48 +CD150 -) (fold-change=1.69±0.18, n=3 for each group). In contrast, mice secondarily transplanted with CD99 KO TET2/NRAS BM exhibited similar engraftment, survival, and morphologic evidence of AML as those transplanted with CD99 WT TET2/NRAS BM. In conclusion, our data demonstrate that LSC function in AML driven by mutated FLT3, but not mutated NRAS, is dependent on maintenance of regulated protein synthesis by CD99. We speculate that this differential dependency may reflect a different cell-of-origin, as recent work has shown that AMLs associated with FLT3-ITD exhibit particularly primitive cellular hierarchies (PMID: 35618837). Finally, our results suggest that certain high-risk AML sub-types associated with elevated CD99 expression may be highly sensitive to strategies disrupting protein synthesis to deplete LSCs.