Subtype Of Acute Myeloid Leukemia Research Articles

Enhancer-activated RET confers protection against oxidative stress to KMT2A-rearranged acute myeloid leukemia.

Ectopic activation of rearranged during transfection (RET) has been reported to facilitate lineage differentiation and cell proliferation in different cytogenetic subtypes of acute myeloid leukemia (AML). Herein, we demonstrate that RET is significantly (p < 0.01) upregulated in AML subtypes containing rearrangements of the lysine methyltransferase 2A gene (KMT2A), commonly referred to as KMT2A-rearranged (KMT2A-r) AML. Integrating multi-epigenomics data, we show that the KMT2A-MLLT3 fusion induces the development of CCCTC-binding (CTCF)-guided de novo extrusion enhancer loop to upregulate RET expression in KMT2A-r AML. Based on the finding that RET expression is tightly correlated with the selective chromatin remodeler and mediator (MED) proteins, we used a small-molecule inhibitor having dual inhibition against RET and MED12-associated cyclin-dependent kinase 8 (CDK8) in KMT2A-r AML cells. Dual inhibition of RET and CDK8 restricted cell proliferation by producing multimodal oxidative stress responses in treated cells. Our data suggest that epigenetically enhanced RET protects KMT2A-r AML cells from oxidative stresses, which could be exploited as a potential therapeutic strategy.

Function of PML-RARA in Acute Promyelocytic Leukemia.

The transformation of acute promyelocytic leukemia (APL) from the most fatal to the most curable subtype of acute myeloid leukemia (AML), with long-term survival exceeding 90%, has represented one of the most exciting successes in hematology and in oncology. APL is a paradigm for oncoprotein-targeted cure.APL is caused by a 15/17 chromosomal translocation which generates the PML-RARA fusion protein and can be cured by the chemotherapy-free approach based on the combination of two therapies targeting PML-RARA: retinoic acid (RA) and arsenic. PML-RARA is the key driver of APL and acts by deregulating transcriptional control, particularly RAR targets involved in self-renewal or myeloid differentiation, also disrupting PML nuclear bodies. PML-RARA mainly acts as a modulator of the expression of specific target genes: genes whose regulatory elements recruit PML-RARA are not uniformly repressed but also may be upregulated or remain unchanged. RA and arsenic trioxide directly target PML-RARA-mediated transcriptional deregulation and protein stability, removing the differentiation block at promyelocytic stage and inducing clinical remission of APL patients.

PIEZO1 is essential for the survival and proliferation of acute myeloid leukemia cells.

Leukemogenesis is a complex process that interconnects tumoral cells with their microenvironment, but the effect of mechanosensing in acute myeloid leukemia (AML) blasts is poorly known. PIEZO1 perceives and transmits the constraints of the environment to human cells by acting as a non-selective calcium channel, but very little is known about its role in leukemogenesis. For the first time, we show that PIEZO1 is preferentially expressed in healthy hematopoietic stem and progenitor cells in human hematopoiesis, and globally overexpressed in AML cells. In AML subtypes, PIEZO1 expression associates with favorable outcomes as better overall (OS) and disease-free survival (DFS). If PIEZO1 is expressed and functional in THP1 leukemic myeloid cell line, its chemical activation doesn't impact the proliferation, differentiation, nor survival of cells. However, the downregulation of PIEZO1 expression dramatically reduces the proliferation and the survival of THP1 cells. We show that PIEZO1 knock-down blocks the cell cycle in G0/G1 phases of AML cells, impairs the DNA damage response pathways, and critically increases cell death by triggering extrinsic apoptosis pathways. Altogether, our results reveal a new role for PIEZO1 mechanosensing in the survival and proliferation of leukemic blasts, which could pave the way for new therapeutic strategies to target AML cells.

Hyperactive Natural Killer cells in Rag2 knockout mice inhibit the development of acute myeloid leukemia

Immunotherapy has attracted considerable attention as a therapeutic strategy for cancers including acute myeloid leukemia (AML). In this study, we found that the development of several aggressive subtypes of AML is slower in Rag2−/− mice despite the lack of B and T lymphocytes, even compared to the immunologically normal C57BL/6 mice. Furthermore, an orally active p53-activating drug shows stronger antileukemia effect on AML in Rag2−/− mice than C57BL/6 mice. Intriguingly, Natural Killer (NK) cells in Rag2−/− mice are increased in number, highly express activation markers, and show increased cytotoxicity to leukemia cells in a coculture assay. B2m depletion that triggers missing-self recognition of NK cells impairs the growth of AML cells in vivo. In contrast, NK cell depletion accelerates AML progression in Rag2−/− mice. Interestingly, immunogenicity of AML keeps changing during tumor evolution, showing a trend that the aggressive AMLs generate through serial transplantations are susceptible to NK cell-mediated tumor suppression in Rag2−/− mice. Thus, we show the critical role of NK cells in suppressing the development of certain subtypes of AML using Rag2−/− mice, which lack functional lymphocytes but have hyperactive NK cells.

Exposure to persistent organic pollutants in newborn dried blood spots and childhood acute myeloid leukemia

Acute myeloid leukemia (AML) is a rare malignancy representing 15–20% of all leukemia diagnoses among children. Maternal exposure to persistent organic pollutants is suggestive of increased risk for childhood AML based on existing evidence. We aimed to evaluate the relationship between persistent organic pollutants and childhood AML using newborn dried bloodspots (DBS) from the Michigan BioTrust for Health. We obtained data on AML cases diagnosed prior to 15 years of age (n = 130) and controls (n = 130) matched to cases on week of birth from the Michigan Department of Health and Human Services. We quantified levels of dichlorodiphenyldichloroethylene (p,p’-DDE), hexachlorobenzene (HCB), and polybrominated diphenyl ether congener 47 (BDE-47) in newborn DBS. We also evaluated other organochlorine pesticides, polychlorinated biphenyls, polybrominated biphenyl congener 153, and polybrominated diphenyl ethers, though these were not further evaluated as >60% of observations were above the limit of detection for these chemicals. To evaluate the association between each chemical and AML, we used multivariable conditional logistic regression. In our multivariable model of HCB adjusted for month of birth, maternal age at delivery, and area poverty, we observed no association with AML (Odds Ratio [OR] per interquartile range increase: 1.17, 95% CI: 0.80, 1.69). For p,p’-DDE, ORs were significantly lower for those exposed to the highest tertile of p,’p-DDE (≥0.29 pg/mL, OR: 0.32, 95% CI: 0.11, 0.95) compared to the first tertile (<0.09 pg/mL). We observed no statistically significant associations between HCB and BDE-47 and AML. We observed a reduced odds of exposure to p,’p-DDE and an increased, though imprecise, odds of exposure to HCB among AML cases compared to controls. Future studies would benefit from a larger sample of AML patients and pooling newborn DBS across multiple states to allow for additional variability in exposures and evaluation of AML subtypes, which may have differing etiology.

C-Kit and Flt3 mutation status in acute myeloid leukaemia with cytogenetic correlation and prognosis: A series of 75 cases

Recent WHO classification (5 edition) has included Acute Myeloid Leukaemia (AML) with mutations in NPM1, CEBPA as separate entities along with AML with cytogenetic abnormalities in AML with recurrent genetic abnormalities. Even though C-kit and FLT3 mutations in AML have no diagnostic importance, prognostic significance in different subtypes of AML for the presence of these mutations are not the same.To assess the correlation between French American British (FAB) AML classification, specific cytogenetic abnormalities with C-kit, and FLT3 mutation in AML and to evaluate the prognosis and survival of AML patients with respect to cytogenetic abnormalities and C-kit and FLT3 mutation status. Retrospectively all AML cases in which C-kit and FLT3 mutation status was assessed were retrieved; C-kit D816V mutation status had been assessed by Real time PCR; For FLT3 mutation, both Internal tandem duplication (ITD) and D835V mutation status had been assessed using PCR and gel electrophoresis; The data regarding morphological with immunophenotypic diagnosis, conventional karyotyping, FISH for translocation 8;21 (t (8;21)) and inversion16 / translocation16;16 (t (16;16) were also retrieved in all these cases along with follow-up from hospital records. Total 75 cases were included; Male/ Female ratio was 1.21:1 (41/34); Median age was 31 (Range: 2 - 64); 18 cases had translocation 8;21 (t (8;21)); 3 cases showed inversion16 / translocation16;16 (t (16;16); Out of the 18 cases which showed t (8;21), 10 cases had associated loss of sex chromosome. Eight cases had C-kit D816V mutation; three of which had t (8;21) while two had inversion 16. 12 cases had FLT3 mutations among which nine were ITD while three had D835V mutation. On karyotyping, one of these cases showed hyperdiploidy while the majority had normal karyotype. A single case had both C-kit D816V mutation and FLT3 D835V mutation with inversion 16 on karyotyping; Most common type of AML in both cases with FLT3 mutation and C-kit mutation was AML-M2 (FAB); Commonest karyotyping abnormality for cases with C-kit mutation was t(8;21); while for FLT3 mutation, the majority had normal karyotype; The single case which had both C-kit D816V mutation and FLT3 D835V mutation was alive event-free at three-year follow-up. Both FLT3 ITD and TKD mutations had a worse prognosis in our study. However, AML cases with C-kit mutation had a similar prognosis comparable to C kit negative cases. A large-scale study is required to elucidate the significance of this.

Panel-based RNA fusion sequencing improves diagnostics of pediatric acute myeloid leukemia

New methods like panel-based RNA fusion sequencing (RNA-FS) promise improved diagnostics in various malignancies. We here analyzed the impact of RNA-FS on the initial diagnostics of 241 cases with pediatric acute myeloid leukemia (AML). We show that, compared to classical cytogenetics (CCG), RNA-FS reliably detected risk-relevant fusion genes in pediatric AML. In addition, RNA-FS strongly improved the detection of cryptic fusion genes like NUP98::NSD1, KMT2A::MLLT10 and CBFA2T3::GLIS2 and thereby resulted in an improved risk stratification in 25 patients (10.4%). Validation of additionally detected non-risk-relevant high confidence fusion calls identified PIM3::BRD1, C22orf34::BRD1, PSPC1::ZMYM2 and ARHGAP26::NR3C1 as common genetic variants and MYB::GATA1 as recurrent aberration, which we here describe in AML subtypes M0 and M7 for the first time. However, it failed to detect rare cytogenetically confirmed fusion events like MNX1::ETV6 and other chromosome 12p-abnormalities. As add-on benefit, the proportion of patients for whom measurable residual disease (MRD) monitoring became possible was increased by RNA-FS from 44.4 to 75.5% as the information on the fusion transcripts’ sequence allowed the design of new MRD assays.

How to avoid early mortality in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL), a phenotypically and genotypically unique subtype of acute myeloid leukemia, has seen unprecedented advances in its management since the introduction of all-trans retinoic acid (ATRA) and arsenic trioxide. However, the phenomenal pharmacologic conversion of this once highly fatal disease to one with a long-term survival exceeding 90% among patients who survive induction remains impaired by the significant incidence of early death (ED) reaching 30% in some real-world studies. The key driver for ED in APL is catastrophic hemorrhage with a proclivity for cranial sites. Most EDs in APL are currently considered preventable. Here, we discuss the concept of early death in APL and its characteristics. Importantly, we outline implementable strategies to reduce the incidence of ED. Early recognition of APL underpins these preventive measures as significant delays in the diagnosis increase the likelihood of ED. While early administration of ATRA is often taught to all hematology trainees, this lifesaving intervention is only possible if providers, including those in emergency departments and urgent/immediate care settings, are trained to have a high index of suspicion and competence to recognize the morphologic and clinical characteristics of the disease. Other proposed strategies tackle the complications that can be present at diagnosis or arise during induction therapy and address the issues of expert consultation and protocol adherence in the management of these patients. While some of these measures appear intuitive and others aspirational, widespread adoption could bring about an era of cure for almost every patient with APL.

Novel immunotherapies in the treatment of AML: is there hope?

The success of allogeneic stem cell transplantation has demonstrated the potential for immunotherapy to treat acute myeloid leukemia (AML). Although alternative T-cell-based immunotherapies have shown efficacy, they also pose the risk of on-target off-leukemia hematotoxicity. So far, adoptive autologous or allogeneic chimeric antigen receptor (CAR) T/natural killer cell therapy is almost exclusively employed as a bridge-to-transplant strategy in the context of clinical trials. For now, clinical trials predominantly target lineage-restricted antigens, but emerging approaches focus on leukemia-associated/specific intracellular target antigens, including dual and split targeting strategies. Adapter CAR T cells and T-cell-recruiting bispecific antibodies offer transient exposure with enhanced safety and multitargeting potential against antigen-escape variants. However, these have yet to demonstrate sustained responses and should be used earlier to treat low leukemia burden, preferably if measurable residual disease is present. To address immune dysregulation and enhance T-cell fitness, novel CAR T and bispecific designs, along with combinatorial strategies, might prove essential. Furthermore, genetic associations with inflammatory bone marrow signatures suggest the need for tailored platforms in defined AML subtypes. The eagerly anticipated results of trials investigating magrolimab, an anti-CD47 antibody targeting the "do not eat me" signal in p53-mutated AML, should shed further light on the potential of these evolving immunotherapeutic approaches.

Transcriptional control of leukemogenesis by the chromatin reader SGF29

AbstractAberrant expression of stem cell–associated genes is a common feature in acute myeloid leukemia (AML) and is linked to leukemic self-renewal and therapy resistance. Using AF10-rearranged leukemia as a prototypical example of the recurrently activated “stemness” network in AML, we screened for chromatin regulators that sustain its expression. We deployed a CRISPR-Cas9 screen with a bespoke domain-focused library and identified several novel chromatin-modifying complexes as regulators of the TALE domain transcription factor MEIS1, a key leukemia stem cell (LSC)–associated gene. CRISPR droplet sequencing revealed that many of these MEIS1 regulators coordinately controlled the transcription of several AML oncogenes. In particular, we identified a novel role for the Tudor-domain–containing chromatin reader protein SGF29 in the transcription of AML oncogenes. Furthermore, SGF29 deletion impaired leukemogenesis in models representative of multiple AML subtypes in multiple AML subtype models. Our studies reveal a novel role for SGF29 as a nononcogenic dependency in AML and identify the SGF29 Tudor domain as an attractive target for drug discovery.

Abstract B032: Outcomes of patients with acute promyelocytic leukemia treated at John Peter Smith Hospital

Abstract Background: Acute Promyelocytic Leukemia (APL) is a subtype of Acute Myeloid Leukemia (AML), accounting for about 10% of all AML cases. Historically, APL was considered to be the subtype of AML with the worst prognosis because most newly diagnosed patients died within a few weeks, and only 35% to 45% of patients achieved long-term survival. The discovery and application of all trans retinoic acid (ATRA) improved complete remission (CR) rate and overall survival (OS) to 70%. Combination of ATRA, a vitamin A derivative, and arsenic trioxide (ATO) as induction therapy for APL resulted in 90-95% CR and &amp;gt;90% 5-year OS. Even though CR and OS has improved, early death (5%-10%) remains the most challenging issue for the disease, especially in resource poor settings. Since APL is rare, oncologists in community may not be fully equipped to treat early complications. John Peter Smith hospital (JPS) is a safety-net hospital serving the needs of underserved population in north Texas. We report outcomes of patients with APL treated at JPS. Methods: Data was obtained from the JPS Oncology Registry. Patients were diagnosed with APL from 1/1/2017 to 12/31/2020 with partial or complete treatment at JPS. We collected non-identifiable patient demographics, diagnosis and initial treatment and complication details. Remission documentation by disappearance of the chromosome abnormality, time from diagnosis to date of death (if applicable) or last activity in medical chart (any phone call with patient, office visit) before 6/12/2023 were obtained. Time to CR and any use of consolidation or maintenance treatment was also collected. Results: 8 APL patients were included- 3 were high-risk patients and 5 were low-risk. Half were male. Age ranged from 22 to 64 years. 5 were under the age of 40 years, 2 were in their 40s and 1 was in 60s at diagnosis. Hispanic:4, Black:3, Asian:1. Induction treatment included ATRA+ATO in 6 patients, while 2 high risk patients received ATRA plus anthracycline-based chemotherapy. Bone marrow morphology post induction was negative in all patients indicating CR (median time to CR was 41.5 days). During induction, treatment complications occurred in all patients including bleeding (75%), clotting (63%), neutropenic fever (50%). Differentiation syndrome (50%) and DIC (50%). All patients underwent ATRA+ATO consolidation treatment for 8-10 months. No patients relapsed. All patients were alive at the time of last medical chart entry (6/12/2023) without active cancer. Conclusions: All patients were in CR post induction treatment. 4 had minimal residual disease post induction. All patients received consolidation with ATRA+ATO, eventually there was no disease detected at molecular level. No patients relapsed. All patients were alive with no active disease at their last follow-up. Excellent outcomes in APL are possible at safety-net setting but will need diligent care to prevent, predict and treat early complications; this may require communication with academic leukemia specialists when transfer to leukemia treatment centers is not possible. Citation Format: Kavya Athipatla, Kari Teigen, Kalyani Narra. Outcomes of patients with acute promyelocytic leukemia treated at John Peter Smith Hospital [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B032.

Abstract A049: Investigation of FHD-609, a potent degrader of BRD9, in preclinical models of acute myeloid leukemia (AML)

Abstract Acute myeloid leukemia (AML) is a complex disease with multiple sub-types, each characterized by unique clinical and molecular features, driving the need to develop targeted therapies which exploit specific vulnerabilities. Bromodomain-containing protein 9 (BRD9) is a component of the ncBAF chromatin remodeling complex, and has been recently indicated as a strong dependency in AML (Weisberg et al 2022). It has been shown that inhibitors of BRD9 induce growth inhibition and expression of apoptotic makers in AML cell lines (Hohmann et al 2016; Zhou et al 2021). It has also been reported that BRD9 is critical to AML cell survival through the maintenance of STAT5 signaling (Del Gaudio et al 2019). Herein, we profile the in vitro anti-proliferative effects of FHD-609, a potent and selective degrader of BRD9, in a panel of 40 AML cell lines representative of a broad range of AML sub-types. We observed that FHD-609 was effective at inhibiting the growth of a subset of AML cell lines, consistent with previous reports in the literature. We further investigated whether treatment of AML cell lines with FHD-609 would induce changes in cell cycle, and found an increase in G1 in the same subset of cell lines that showed cell growth inhibition. Furthermore, treatment with FHD-609 induced apoptosis in these cell lines. To identify the mechanisms of action of FHD-609 in AML, as well as potential predictive biomarkers for AML sensitivity to FHD-609, we performed a range of mechanistic studies including ATACseq, ChIPseq and RNAseq in AML cells treated with FHD-609. We observed significant changes in the chromatin landscape in sensitive AML cell lines, and negligible changes in insensitive cell lines. Further bioinformatics analysis identified a possible biomarker strategy that correlates with the sensitivity of AML cell lines to FHD-609 in vitro. To confirm whether this biomarker strategy would successfully predict response in vivo, we dosed AML CDX and PDX models with FHD-609 based on the biomarker selection strategy. We observed a strong anti-tumor effect in AML CDX models and significantly extended survival of the mice in the AML PDX models. In summary, we have shown that FHD-609 demonstrates strong anti-tumor efficacy in a subtype of AML and have identified a possible biomarker strategy to predict response. Citation Format: Claudia Dominici, David Mayhew, Ammar Adam, Flore Uzan, Victoria Garbitt-Amaral, Oliver Mikse, Brandon Antonakos, Hafiz Ahmad, Salonee Parikh, Mei Yun Lin, Gabriel Sandoval, David Lahr, Hsin-Jung Wu, Mengni Xu, Sean Brennan, Luis M. M. Soares, Jordana Muwanguzi, Huawei Chen, Zhaoxia Yang, Jason T Lowe, Matt Netherton, Laura Zawadzke, Johannes Voigt, Liyue Huang, Sabine Ruppel, Ho Man Chan, Ryan Kruger, David S Milan, Scott Innis, Qianhe Zhou, Steven F Bellon. Investigation of FHD-609, a potent degrader of BRD9, in preclinical models of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A049.

Abstract C099: Establishment and characterization of an NUP98-KDM5A-driven AML XPDX model

Abstract Background: Acute myeloid leukemia (AML) with NUP98 fusions is associated with poor prognosis in adults and especially children. Pediatric AML is a rare and heterogeneous disease characterized by a high prevalence of gene fusions as driver mutations, including the NUP98-KDM5A fusion found in a particular subgroup of patients with complex karyotypes. To better understand this subtype of AML, we established and characterized a XPDX model designated ST1653 representing NUP98-KDM5A-driven disease from a previously treated AML patient. We performed DNA and RNA-based sequencing of the model. Further we evaluated in vivo sensitivity towards cytarabine and inhibitors targeting the JAK and CDK6 pathways, which have been shown to be important in NUP98-KDM5A-driven AML. Methods: ST1653 was established from a bone marrow aspirate collected from a 63-year-old Caucasian male who had AML recurrence following high dose cytarabine. The sample was injected subcutaneously into a triple-deficient mouse and the P0 model passaged after seventy days into CB-17 SCID and athymic nude mice and further developed until stable. WES and RNAseq were then performed. For in vivo studies, ST1653 was evaluated against cytarabine and the JAK inhibitors ruxolitinib, baricitinib and tofacitinib and the CDK4/6 inhibitors palbociclib, abemaciclib and ribociclib. Endpoints in all studies included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C&amp;lt;0%) versus Day 0 tumor volume was also reported. Results: Growth of the ST1653 AML XPDX model was similar in CB-17 SCID or athymic nude mice with a median time to endpoint of forty-five days, although the take rate in the SCID strain was superior. Sequencing identified the presence of the NUP98-KDM5A fusion and chimeric protein expression was confirmed. In vivo, ST1653 was insensitive to cytarabine and the JAK inhibitors but demonstrated limited sensitivity to CDK4/6i treatment. Conclusion: We have established and characterized a XPDX model of NUP98-KDM5A-driven AML including sequencing and in vivo evaluation. This model is an important tool for developing novel therapies to this high-risk leukemia subtype. Citation Format: Armando Diaz III, Alyssa Simonson, Johnnie Flores, Joseph Holahan, Kyriakos P Papadopoulos, Jim Lund, Drew Rasco, Amita Patnaik, Michael J Wick. Establishment and characterization of an NUP98-KDM5A-driven AML XPDX model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C099.

Data-Driven Multiparameter Flow Cytometry (MFC) Classification of Blast Differentiation in Elderly Patients with Acute Myeloid Leukemia (AML)

Background: The identification of the nearest normal counterpart of blasts has been part of the patients' diagnostic workup and contributed to the sub-classification of AML. While there is a progressive displacement of morphologically defined AML subtypes by those with recurrent genetic abnormalities, recent studies in younger patients eligible for intensive chemotherapy suggested that a phenotypic sub-classification of AML might be prognostic. However, an antigen-based assessment of blast differentiation is in part complex and subjective, and to our knowledge has not been systematically investigated in elderly AML. Aim: Develop a data-driven approach to facilitate and systematize the phenotypic dissection of older AML patients ineligible to receive intensive chemotherapy. Methods: The study included 252 patients (&amp;gt;65yo) that were randomized to azacitidine vs low-dose Ara-C plus fludarabine in the PETHEMA-FLUGAZA trial. MFC was performed using the EuroFlow AML panel in bone marrow aspirates. The percentage of expression of each marker in hematopoietic progenitor cells (HPC), granulocytic, monocytic and erythroid precursors, as well as in maturing granulocytes, monocytes and nucleated red blood cells (NRBC) was determined in 15 healthy adults. The data were merged and a matrix was obtained for each subject. The percentage of expression of each marker was determined in patients' blasts. After projecting each case onto the matrix generated from healthy adults, the distance between blasts and normal populations was calculated, and the shortest distance identified the corresponding differentiation stage. Blasts were isolated according to patient-specific aberrant phenotypes for DNA and RNA co-isolation and subsequent next-generation sequencing (n=179/252) and RNA-seq (n=190/252). Results: Of the 252 patients, 64% were classified with an HPC-like AML phenotype, 27% a granulocytic-precursor, 2% monocytic-precursor, 2% erythroid-precursors, &amp;lt;1% maturing-granulocytic, 4% maturing-monocytic and &amp;lt;1% maturing-NRBC. Cell types from the same lineage were regrouped into granulocytic-, monocytic and erythroid-like AML. Overall, there was a 46% concordance between phenotypic and FAB classification. Interestingly, patients classified as HPC-like AML treated with azacitidine (and not those receiving semi-intensive chemotherapy) showed slightly longer overall survival when compared to those with maturing AML subtypes. We next investigated the relationship between phenotypically-defined stages of blast arrest and the presence of underlying mutations. Interestingly, there was scarce overlap in the top three recurrently mutated genes across the HPC- ( SRSF2, TET2, RUNX1), granulocytic- ( ASXL1, FLT3, TET2), monocytic ( FLT3, NF1, TET2), and erythroid-like ( TP53, DNMT3A, NF1) AML subtypes. Conversely, EPAS1, FLT3 and NF1 were more frequently mutated ( p&amp;lt;.05) in erythroid- and monocytic- vs HPC-like AML. Amongst the maturing subtypes, monocytic-like AML was characterized by frequent SH2B3 and absent TP53 mutations ( p&amp;lt;.05). The comparison between HPC-like vs all maturing subtypes identified several deregulated genes associated with different stages of hematopoiesis (i.e., HOXA3, ANKRD28, CLEC2B, HSD17B11, SYPL1, HBD, S100A8, ITGAM). Granulocytic-like AML patients showed deregulation of 200 genes; 44 of which were upregulated and enriched in granulocytic-related pathways. Only 23 genes were deregulated upon comparing the transcriptional profile of HPC- vs monocytic-like blasts. A total of 415 genes were deregulated between HPC- and erythroid-like blats; 269 of which being overexpressed and associated with pathways such as transferrin transport, erythrocyte development and differentiation. Accordingly, the transcriptional profile of blasts corroborated our data-driven approach. Conclusions: This study builds upon recent interest in non-genetic drivers of AML heterogeneity and classification systems based on the partial recapitulation of myeloid development, and shows that these concepts may be applied in elderly patients. Our proof-of-concept data-driven approach has the potential to be incorporated into flow cytometry analytical software towards a more objective dissection of AML subtypes based on blast differentiation, which could potentially yield prognostic information complementary to other routine diagnostic procedures.

Co-Occurring Mutations in ASXL1 and SRSF2 Define a Unique, Prognostically Relevant Chromatin-Spliceosome Gene Signature in De Novo acute Myeloid Leukemia

Background: Prior studies with extensive acute myeloid leukemia (AML) molecular profiling identified various subgroups wherein co-mutations in genes related to chromatin regulation and RNA splicing showed no survivors, irrespective of age or treatment. Within the chromatin-spliceosome family, co-mutations of ASXL1 and SRSF2 are linked to poor outcomes. However, the representation of this co-mutational pair in de novo, secondary, and therapy-associated AML is not well described. Also, there is no evidence to demonstrate if HSCT would alter poor prognostic outcomes in this unique molecular subgroup of AML. We sought to assess the clinicogenomic characteristics of ASXL1/ SRSF2 co-mutation and its outcomes in a large, well-annotated, sequenced cohort of real-world AML patients. Methods: We conducted a retrospective analysis of adult AML patients with ASXL1 MT/ SRSF2 MT by screening well-annotated public databases, cBioPortal (Cerami et al., 2012) and AACR GENIE (v 13.1), and expanded with a published metanalytic cohort of various sub-studies from Cleveland Clinic Foundation from 2012-2021 (Awada et al., Blood 2021, Kewan et al., Nature Communications, 2023). We analyzed the NGS data in the context of baseline clinical parameters, coexisting mutations, variant allele frequencies, karyotype, and AML subtypes. The primary endpoint was overall survival (OS), and secondary outcomes included survival based on AML subtype, karyotype, and hematopoietic stem cell transplant treatment (HSCT). The chi-square test was used to study various described parameters, and Kaplan-Meir curves were used for survival analyses. Results: We screened a total of 15,742 adult patients with AML, and 2,471 eligible patients were separated into 3 cohorts: 280 (11.3%) patients with ASXLMT/SRSF2 MT,1,025 (41.5%) patients with ASXLMT/ SRSF2WT and 1,166 (47.2%) patients with ASXLWT/ SRSF2MT. In addition, three AML subtypes were established: de novo AML (dnAML), secondary AML (sAML), and therapy-associated AML (tAML). The median age of the co-mutated cohort was 70.1 ys. [range 42-100 ys.], compared to the ASXL1MT [17-100 ys., p=0.0046] and SRSF2MT cohorts [25-99 ys., p=0.5]. Females were more prominently represented in the ASXL1MT cohort compared to the SRSF2MT cohort [47% vs 30%, p&amp;lt;0.0001]. All three cohorts were predominantly enriched by dnAML (69%, 70%, and 67%), the ASXL1MT cohort had the most tAML (2.2%), and sAML enriched the SRSF2MT cohort (31%). Abnormal karyotype enriched the ASXL1MT/ SRSF2WT cohort compared to SRSF2MT [50% vs 39%, p=0.0001] and equivalent in ASXL1MT cohorts (50% vs 51%, p=0.06]. Median OS was driven in part by the ASXL1MT clone and lowest for the co-mutated compared to the ASXL1MT [12.0 vs 12.5 mo., p=0.0055] and SRSF2MT cohorts [12.0 vs 17.5 mo., p =&amp;lt;0.0001]. When divided by AML-subtype, dnAML had the lowest median OS in the co-mutated, compared to the ASXL1MT [12 vs 15 mo., p =0.03] and the SRSF2MT cohorts [12 vs 17 mo., p =0.02]. sAML had nearly equivalent survival across all cohorts (10.0 vs 9.3 vs 9.4 mo.), and although tAML offered numerically better survival (NE vs 8.7 vs 17.5 mo.), it was not significant. Normal karyotype offered a numerical survival advantage (NE vs 14.39 vs 16.85 mo.). The median number of mutations in the co-mutated cohort was 4 (range 3 - 7), Fig 1(A). The most common co-occurring mutations in this cohort were RUNX1 (78%), TET2 (48%), IDH2 (43%), NRAS (16%), and CEBPA (15%). 21 patients were transplant-eligible from the co-mutated cohort, with median OS after HSCT being 33 vs 12 mo. without HCT (p =0.0010), Fig 1 (B). Conclusions: ASXL1/ SRSF2 co-mutated patients represent a unique chromatin-spliceosome signature, most prominently in de novo AML, associated with a significantly lower OS. Therefore, it must be identified at diagnosis for improved prognostic assessment and potential therapeutic implications. HSCT can provide longer-term survival in these patients. This represents the largest reported cohort of patients with ASXL1/ SRSF2 co-mutated AML. Further studies are warranted to validate the chromatin-spliceosome signature and prognosis.

Elucidating Single-Cell Transcriptional Differences of the MLL Subtype in Pediatric Acute Myeloid Leukemia Using Machine Learning and Gene Regulatory Analysis

Introduction: The prognosis for highly heterogeneous pediatric acute myeloid leukemia (AML), classified into eight major subtypes, varies considerably. Common pediatric AML abnormalities include the ones involving mixed lineage leukemia (MLL) gene rearrangements, RUNX1/RUNX1T1 (RUNX1) fusion, and chromosome 16 (inv(16)) inversion. MLL has the worst prognosis (5-year OS: 47%) while RUNX1 and inv(16) have much better prognoses (5-year OS: 91% and 87% respectively) ( von Neuhoff et al., 2010). Using single-cell profiling, our group had developed gene signatures to discriminate between these three subtypes, with the identified genes in the MLL signature correlating with a worse prognosis ( Krishnan et al., 2022). Here, to further improve the robustness of the blast-associated signatures, we employed a machine learning model and included more healthy control samples. Revealing differences between these subtypes' immune microenvironments (IME) is critical for identifying the cellular/molecular drivers of the poorer prognosis associated with the MLL subtype. To identify subtype-specific differences in genes/pathways, we performed an in-depth analysis of the blasts and IME. Methods: We analyzed single-cell transcriptome data from pediatric AML bone marrow (BM) samples collected at diagnosis (MLL, n=4; RUNX1, n=4; inv(16), n=3) and healthy BM samples (n=9) ( Bailur et al., 2020; Caron et al., 2020). Quality control and filtering yielded &amp;gt;63,000 cells. The 7-gene signature from Thomas et al. (2020) was used to distinguish leukemic blasts from non-blast cells. Differential expression (DE) analysis was conducted using Wilcoxon's test between the three subtypes' blast cells and healthy myeloid cells (&amp;gt;50% subtype-blasts expression, &amp;lt;20% other samples' expression). The most differentially expressed genes (Log2FC&amp;gt;0.6, p&amp;lt;1e-5) were used to derive the subtype-specific gene signatures, and they were then further refined by considering only the genes that were also differentially expressed (p&amp;lt;1e-5) in the TARGET-AML bulk RNA-seq dataset ( McNeer et al., 2019). These signatures were validated by training a machine-learning-based LightGBM classifier ( Ke et al., 2017) on the gene expression levels in additional TARGET bulk RNA-seq data to differentiate between the three subtypes by themselves as well as other AML and healthy samples. Cell communication and gene regulatory analysis were performed using the CellChat ( Jin et al., 2021) and pySCENIC ( Aibar et al., 2017) tools respectively. Results: Our analysis confirmed overexpression of the previously reported SOCS2, KCNE5 and TRPM4 genes in MLL leukemic blasts and identified new markers including LAMP5, LTC4S and HOXA9. HOXA9 is a transcription factor that has been shown to result in a poorer prognosis for patients suffering from AML ( Collins et al., 2015). CAV1 was found to be overexpressed in RUNX1-subtype blasts in addition to the POU4F1, NPW and TRH genes found previously. The inv(16) blast signature comprised of the AUTS2, PAM, ST18, SPARC and TSC22D1 genes. The LightGBM classifier trained on these signatures exhibited an overall AUROC test score of 0.9996 when discriminating between just the three subtypes and 0.9651 when other AML subtypes and healthy controls were considered as well (Fig. 1A). In the T-cell compartment of the IME, MLL patients not only had a significantly (p&amp;lt;1e-5) lower proportion of CD8+ Naïve T-cells than any of the other subtypes but also had a significantly (p=0.026) smaller overall proportion of CD8+ T-cells (Fig. 1B). Gene regulatory analysis revealed that MLL leukemic blasts had significantly (p&amp;lt;2e-16) enriched activity of the SPI-B transcription factor (TF), known to suppress CD8+ T-cell proliferation in various cancers ( Huang et al., 2021). Additionally, cell communication analysis revealed significantly (p&amp;lt;0.01) less communication involving the LCK pathway within CD8+ Naïve T-cells, a pathway known to play a key role in proliferation ( Seddon et al., 2000) Conclusions: This study evaluated the differences between the MLL, RUNX1, and inv(16) AML subtypes at the single-cell level, discovered gene signatures that effectively discriminate between the three subtypes as well as other AML and healthy samples, and revealed that the poorer prognosis of the MLL subtype may be attributable to dysregulation of CD8+ T-cell proliferation caused by abnormalities in the SPI-B TF and the LCK signaling pathway.

SETDB1 Suppresses Interferon Responses and NK Cell-Mediated Immunosurveillance Specifically in Monocytic AML

Acute myeloid leukemia (AML) with monocytic differentiation (monocytic AML) responds poorly to current treatments, including venetoclax-based therapy. To identify novel therapeutic targets in monocytic AML, we conducted in vivo and in vitro CRISPR/Cas9 library screens using a pooled sgRNA library targeting epigenetic regulators with a mouse monocytic AML model driven by SETBP1 and ASXL1 mutations (cSAM: combined expression of SETBP1 and ASXL1 Mutations). We ranked all the epigenetic genes according to the in vivo specific essentiality, and selected several candidates which were more depleted in mice rather than in cell cultures. The top-ranked gene was Setdb1, which encodes a histone H3 lysine 9 (H3K9) methyltransferase. In addition, several H3K9 trimethylation (H3K9me3) regulators, ATF7IP and TRIM33, were also more important in vivo than in vitro in our screens. ATF7IP is a cofactor of SETDB1 to enhance its enzymatic activity. TRIM33 is also involved in H3K9me3 modification. These data suggest the critical role of the SETDB1-ATF7IP-TRIM33 H3K9 methyltransferase complex in the development of AML in vivo. To validate the results of our screenings and to investigate the role of immune cells in our transplantation assay, we next transplanted control and Setdb1-knockout (KO) cSAM cells into three types of mice with different immune systems: the immunocompetent C57BL/6 mice, the immunodeficient NSG mice lacking T, B and natural killer (NK) cells, and the C57BL/6J- Rag2-/- mice lacking mature T and B cells but possessing NK cells. Although Setdb1 depletion did not inhibit the growth of cSAM cells in NSG mice, it showed the strong growth-inhibitory effect in C57BL/6 mice and Rag2-/- mice (Fig 1A). Since both C57BL/6 mice and Rag2-/- mice have NK cells, these data suggest that SETDB1 promotes leukemogenesis by suppressing NK cell-mediated cytotoxicity in vivo. Mechanistically, loss of Setdb1 induced upregulation of NKG2D ligands, interferon-stimulated genes (ISGs), and endogenous retrovirus (ERV) sequences through H3K9me3 reduction, thereby rendering cSAM cells more susceptible to NK cell-mediated cytotoxicity. Setdb1 depletion also induced myeloid maturation of cSAM cells towards macrophages and downregulation of major histocompatibility complex class I (MHC-I), which contributed to the increased susceptibility of Setdb1-depleted cSAM cells to NK cell-mediated cytotoxicity. Next, we investigated whether SETDB1 inactivation also sensitizes human AML cells to NK cells using the co-culture assay with an IL-2 dependent NK cell line NK-92. Loss of SETDB1 rendered monocytic AML cell lines, NOMO-1, THP-1, OCI-AML3, U937, and NB4, more susceptible to NK-92 cell-mediated cytotoxicity. SETDB1 depletion also induced overactivation of interferon signaling and intrinsic apoptosis in these monocytic AML cells. In contrast, non-monocytic AML cells, such as K562, TF-1, and Kasumi-1, were not sensitive to SETDB1 depletion. SETDB1 depletion neither increased the susceptibility to NK cell-mediated cytotoxicity nor induced apoptosis in non-monocytic AML cells. Subsequent analyses revealed that SETDB1 depletion reduced H3K9me3 at the monocyte-specific enhancer regions with ERV sequences to upregulate NK cell-activating ligands and ISGs. Because the “ERV enhancers” are active only in monocyte lineage, SETDB1 is essential specifically in monocytic AML. Finally, we identified MNDA and its mouse counterpart Ifi203 as biomarkers to predict the sensitivity of each AML to SETDB1 depletion. MNDA is one of the ISGs and is involved in myeloid cell differentiation. Human monocytic AML cells that are dependent on SETDB1 expressed higher MNDA at both RNA and protein levels compared to other subtypes of AML. We also found that Ifi203-high mouse AML cells transformed by MLL-AF9 were sensitive to Setdb1 depletion, whereas Setdb1 was dispensable in Ifi203-low mouse AML cells transformed by RUNX1-RUNX1T1. In summary, our findings reveal the critical and selective role of SETDB1 in monocytic AML. SETDB1 mediates H3K9me3 at the monocyte-specific ERV enhancers to repress the expression of NK cell-activating ligands and ISGs, thereby suppressing immunogenicity and apoptosis in monocytic AML (Fig 1B). SETDB1 as well as its binding partners could be promising therapeutic targets to treat monocytic AMLs that are relatively resistant to the current standard therapies.

Research on Different Compound Combinations of Realgar-Indigo Naturalis Formula to Reverse APL Arsenic Resistance By Regulating Autophagy through mTOR Pathway

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia, and 90% of APL patients carry positive fusion gene of the promyelocytic leukemia protein (PML)- retinoic acid receptor α (RARα), which drives the development of APL. All-trans retinoic acid and arsenic trioxide is able to cure 90% of APL patients by targeting PML-RARα. However, there are still 5%-10% of patients who will relapse due to drug resistance. Compound realgar-indigo naturalis formula (RIF) was developed based on the characteristics of APL pathogenesis. It contains four Chinese herbs: realgar, natural indigo, danshen root and heterophylly falsestarwort root. Clinical studies have confirmed that RIF has a certain efficacy on APL, which shown non-inferiority compared with arsenic trioxide. In addition, RIF combined with all-trans retinoic acid has become the first-line treatment regimen in APL guidelines in China. This study further clarified the efficacy of RIF on arsenic-resistant APL, explored the mechanism, and laid the foundation for further improving the efficacy of APL. O bjective: To investigate the mechanism of different combinations of the compound of RIF in reversing arsenic resistance in APL. M ethods: The arsenic-resistant cell line HL60-PML A216V-RARα, which could stably express PML A216V-RARα, was established by lentiviral transfection. Among compounds of RIF, the active compound of realgar is tetra-arsenic tetra-sulfide (As 4S 4, A), for natural indigo is indirubin (I), for danshen root is tanshinone IIa (T), and the for heterophylly falsestarwort root is total saponins of Radix Pseudostellariae (S). The four compounds were applied on the HL60-PML A216V-RARα cell line. Cell viability was detected by CCK-8 method. Hoechst staining was used to observe cell morphology, Annexin V/PI double staining was applied to detect cell apoptosis, JC-1 detection was used to detect mitochondrial membrane potential, flow cytometry was used to detect CD33 expression to evaluate cell differentiation, and Western blotting was used to observe the changes in the levels of PML A216V-RARα, apoptosis-related factors, PI3K/AKT/mTOR and autophagy in the control, A, ITS and AITS groups. The PI3K inhibitor LY294002 and the autophagy inhibitor Baf A1 were combined and applied to observe the changes in the levels of PML A216V-RARα, mTOR and p62 after blocking the PI3K or autophagy pathway. Results: The arsenic-resistant cell line HL60-PML A216V-RARα was successfully established. Compared with the control group, A, ITS and AITS groups all inhibited the growth of HL60-PML A216V-RARα cells in a time-dependent manner ( P &amp;lt; 0.0001). The IC50 value of AITS was significantly lower than the other groups ( P &amp;lt; 0.0001), and AITS group exhibited the strongest inhibitory effect, followed by ITS and A. Hoechst staining and JC-1 detection both showed that AITS had the strongest effect in inducing apoptosis. Annxin V/PI detection showed that the apoptosis rate of the control group was 10.04%, 15.19% for A group, 12.46% for ITS group, and 28.05% for AITS group. The differences between the groups were statistically significant ( P &amp;lt; 0.0001). Detection of CD33 expression showed: control group (79.72%), ITS group (79.19%), A group (28.60%), AITS group (24.46%), and there were significant differences between groups ( P &amp;lt; 0.001 or P &amp;lt; 0.0001). AITS could reduce PML A216V-RARα protein, caspase3, Bcl-2 levels more than ITS and A ( P &amp;lt; 0.05 or P &amp;lt; 0.0001), and ITS was better than AITS and A in increasing cleaved PARP-1 levels ( P &amp;lt; 0.0001). AITS decreased the levels of PI3K, mTOR, P-mTOR, AKT, p-AKT and p62 and increased the level of LC3B ( P &amp;lt; 0.001 or P &amp;lt; 0.0001). Electron microscopic detection of autophagy showed that AITS had the highest level of autophagy, AITS combined with LY294002 increased the level of p62 and decreased the level of mTOR ( P &amp;lt; 0.0001) compared to the AITS group; while when AITS was combined with Baf A1, the level of p62 increased ( P &amp;lt; 0.0001) and the level of mTOR also increased ( P &amp;lt; 0.001). Autophagy could induced by serum starvation. As the concentration of fetal bovine serum decreased, the effects of degrading PML A216V-RARα and p62, and LC3B level increasing were stronger. C onclusion: Tanshinone IIa, indirubin and total saponins of Radix Pseudostellariae could enhance the effect of tetra-arsenic tetra-sulfide down-regulating PML A216V-RARα, and the mechanism was suggested to be related to inhibiting mTOR pathway to activate autophagy.

Characterization of Comparative Clinical Outcomes and Molecular Features of Black Patients with Acute Promyelocytic Leukemia (APL) Reveals Similar Survival and Distinct Genomic Profiles

Introduction: Although APL, a subtype of acute myeloid leukemia (AML), is an aggressive disease, its treatment was revolutionized with combination all- trans retinoic acid (ATRA) and arsenic trioxide (ATO), making APL a highly curable malignancy. Molecularly, APL is characterized by t(15;17) resulting in PML:: RARA gene fusion as disease-initiating event and by several recurrent gene mutations (eg, FLT3) identified in previous studies. In AML, prior studies have established inferior overall survival (OS) of Black versus White AML patients (pts) in both population-based analyses and clinical trials, found a negative prognostic impact of higher social deprivation (SDI), and characterized differences in frequencies and impact of disease-associated gene mutations. Herein, we compared OS of Black and White APL pts and evaluated genomic landscape of Black pts, which has not been comprehensively assessed to date. Methods: For nationwide population analysis, 2 sets of cancer registries were included in the Surveillance Epidemiology End Results (SEER) Program of the National Cancer Institute to identify 829 adults diagnosed with de novo APL (confirmed by cytogenetic and/or molecular findings) in 1995-2019. To assess survival in the setting of clinical trials, we analyzed 249 pts treated on historic frontline Cancer and Leukemia Group B (CALGB)/Alliance for Clinical Trials in Oncology (Alliance) trials with or without inclusion of ATRA (CALGB 9191, 1992-1995; CALGB 9710, 1999-2005). For Alliance pts, neighborhood SDI was assigned based on pt-reported residence zip code and classified as low (1-25, n=33) or high deprivation (26-100, n=95). OS was analyzed separately for pts diagnosed before and after 2012 to account for treatment advances with ATRA/ATO. To assess molecular features, we performed integrated genomic profiling (paired tumor/normal whole-exome and transcriptome sequencing) on 31 Black APL Alliance pts (9/31 completed, with complete results to be presented at the Annual Meeting). Results: On a population level using SEER data, 3-year (3y) OS significantly improved from 65% for cases diagnosed in 1995-2012 to 75% in those diagnosed after 2012 (p=&amp;lt;0.001), concordant with ATRA/ATO use. Notably, OS of Black and White pts did not differ significantly before or after 2012. This held true in race-specific findings for median household income, Yost index and urban/rural status; none of which significantly associated with OS. Older age was the only parameter associated with OS (3y rates, 1995-2012: &amp;lt;40y, 79%; 40-59y, 71%; ≥60y, 44%; and 2012-2019: &amp;lt;40y, 92%; 40-59y, 80%; ≥60y, 56%). Again, OS of Black and White pts did not differ within age groups. Likewise, OS of Black and White APL pts treated in a relatively controlled setting of clinical trials was not significantly different. As in population-based findings, OS improved over time and with ATRA addition (3y OS rates, 1992-1995, 66%; 1999-2005, 85%), and older age was a strong negative OS predictor (p&amp;lt;0.001). When comparing OS of pts treated on clinical trials with population-based OS, trial pts had a longer 3y OS (84% vs 64%, p&amp;lt;0.001). Notably, pts with SDI &amp;gt;25 (n=95) had a worse OS than pts with SDI &amp;lt;25 (n=33, p=0.03). On a molecular level, preliminary results of whole-exome sequencing (finalized for 9/31 Black pts) showed frequent mutations in FLT3 (5/9 pts), a known APL-associated event, but also revealed several recurrent gene mutations not reported in prior APL sequencing efforts, which were almost exclusively based on White pts, including mutations in DPP6, GOLA6L, NLGN2 and SP8 (found in ≥2 pts). Intriguingly, CALR mutations were found in 3/9 pts. Mutations in FLT3 and CALR were mutually exclusive and found in 8/9 pts, with the remaining pt harboring a SORBS2 mutation. Conclusions: In contrast to most cancer types, including AML, there was no OS disparity with respect to race in APL pts, both in population-based analyses and in the setting of historic clinical trials. Older age and residence with high SDI (&amp;gt;25) were negative OS predictors. Molecularly, our pilot data reveal existence of ancestry-associated differences in driver mutations, suggesting that FLT3 and CALR mutations represent necessary proliferative signals in addition to PML:: RARA-driven differentiation arrest. Support: U10CA180821, U10CA180882, U24CA196171; Clinicaltrials.gov IDs: NCT00048958, NCT00899223, NCT00900224; https://acknowledgments.alliancefound.org

Clinical Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation Using Busulfan/Cyclophosphamide and Melphalan or Thiotepa As Conditioning Regimen in 37 Patients with Acute Megakaryoblastic Leukemia

Background Acute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML), which accounts for approximately 10% of childhood AML and about 1% of adult AML. Patients with non-Down's syndrome AMKL (non-DS-AMKL) are often associated with a poor prognosis with a 3-year overall survival (OS) rate of less than 40%. Currently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential curative therapy for AMKL. However, the long-term survival post-transplant needs to be improved. Here we conducted a single-center study to evaluate the long-term efficacy and safety of allo-HSCT in 37 non-DS-AMKL patients using an innovative conditioning regimen of Busulfan/Cyclophosphamide (Bu/Cy) and Melphalan (MEL) or Thiotepa (TT). Methods From August 2015 to July 2023, a total of 37 non-DS-AMKL patients (M/F 21:16) were treated consecutively in our hospital (Hebei Yanda Lu Daopei Hospital). The median age was 2 years (range 1-9 years). Chromosomal abnormalities were present in 24 cases, with fusion genes as follows CBFA2T3/GLIS2 (5 cases) 13.5%, NUP98-KDM5A (2 cases) 5.4%, MLL-AF10 (3 cases) 8.1%, MLL-AF9 (1 case) 2.7%. Gene mutations: WT1 (10 cases) 27%, JAK2 (7 cases) 18.9%, EVI1 (6 cases) 16.2%, TET2 (4 cases) 10.8%, ASXL1 (4 cases) 10.8%, PTPN11 (4 cases) 10.8%, NRAS (3 cases) 8.1%, GATA1 (2 cases) 5.4%. Before transplantation, in bone marrow (BM), 29 patients were in complete remission (CR), 3 were in partial remission (PR) and the rest 5 were in non-remission (NR). Thirty-three patients received the haploidentical transplantation (donors from parents n=31, siblings n=2) including 1 with the 3 rd transplant, and 4 received matched-unrelated donor transplantation. The stem cell sources were from BM and peripheral blood stem cells (PBSCs) among the 33 related donors, and PBSCs for the 4 unrelated donors. Patients received a median cell number of total mononuclear cells 20.15×10 8/kg (10.7-32.85), CD34+ cells 12×10 6/kg(4.05-27.14) and CD3+ cells 4.66×10 8/kg(1.81-19.38). The conditioning regimens administered were modified Bu/Cy (fludarabine 30 mg/m 2/d×5d; cytarabine 2g/m 2/d×5d; Bu 3.2 mg/kg/d×4d, Cy 1.8g/m 2/d×2d; etoposide 100mg/m 2/d×2d; antithymocyte globulin 1.5mg/kg/d×5d) in 2 patients, Bu/Cy+MEL (MEL 55mg/m 2/d×2d) in 31 patients, and Bu/Cy+TT (TT 2.5mg/kg/d×2d) in the rest 4 patients. Cyclosporine A (CSA)+ mycophenolate mofetil (MMF)+ short methotrexate (sMTX) were used as graft-versus-host disease (GVHD) prophylaxis. Results The median follow-up time was 24 months (2-84 months), and all 37 cases survived the graft, with a median leukocyte engraftment of +13 days (9-21 days), and a median platelet engraftment of +9 days (4-38 days). BM was assessed one month after transplantation, with all grafts fully donor-type. The 5-year OS was 54.1% (Figure 1). There was a significantly higher OS in the CR group (5-year OS 69%) than the PR or NR groups (P&amp;lt;0.001) (Figure 2). Among the CR patients, no significant difference was observed in the 3 different condition regimen groups with modified BU/CY group OS of 50%, BU/CY+MEL group OS of 69.6% and BU/CY+TT group OS of 75% (P=0.869) (Figure 3). After transplantation, 17 patients died including 11 from relapse, 4 from GVHD, 1 from gastrointestinal bleeding and 1from cerebral hemorrhage. The cumulative recurrence rate (CIR) was 29.7%, among which the CIR of the CR group was 17.2%, the PR group of 66.7% and the NR group of 80% (P&amp;lt;0.001). Post-transplant, the incidence of acute GVHD (aGVHD) was 35.13% (Grade II-IV), and severe aGVHD (Grade III-IV) incidence was 18.91%. And 51.35% of patients developed chronic GVHD. Other complication incidences were cystitis at 16.21%, cytomegalovirus at 5.94%, Epstein-Barr viremia at 13.5% and infections at 21.62%. Conclusions Our study indicates that the use of modified Bu/Cy +MEL or +TT conditioning regimens in allo-HSCT procedures presents a feasible and effective approach, demonstrating an improvement in overall survival for non-Down Syndrome AMKL patients. Crucially, it was observed that achieving CR prior to transplantation significantly contributes to better outcomes. Therefore, it is strongly recommended that allo-HSCT is performed in CR status. However, to corroborate these findings, it is essential to conduct long-term, large-scale clinical research across multiple centers.