Acute Myeloid Leukemia Phenotype Research Articles

PTPN11 Mutation Clonal Hierarchy in Acute Myeloid Leukemia.

Mutations in protein tyrosine phosphatase non-receptor type 11 ( PTPN11 ) have been considered late acquired mutations in acute myeloid leukemia (AML) development. To interrogate the ontogeny of PTPN11 mutations, we utilized single-cell DNA sequencing and identified that PTPN11 mutations can occur as initiating events in some AML patients when accompanied by strong oncogenic drivers, commonly NPM1 mutations. The co-driver role of PTPN11 mutations was confirmed in a novel murine model that exhibits an AML phenotype with early expansion of a diverse set of variably differentiated myeloid cells that engrafted into immunodeficient and immunocompetent mice. This immune diversity was reconstituted from early precursor cells when engrafted into immunodeficient mice. Moreover, immune diversity was also observed in the blast component of patient samples with NPM1 and PTPN11 mutations, providing novel antigen targets for immune based approaches in this subset of AML that is resistant to multiple targeted therapies.

Microenvironment matters: In vitro 3D bone marrow niches differentially modulate survival, phenotype and drug responses of acute myeloid leukemia (AML) cells

Acute myeloid leukemia (AML) is a deadly form of leukemia with ineffective traditional treatment and frequent chemoresistance-associated relapse. Personalized drug screening holds promise in identifying optimal regimen, nevertheless, primary AML cells undergo spontaneous apoptosis during cultures, invalidating the drug screening results. Here, we reconstitute a 3D osteogenic niche (3DON) mimicking that in bone marrow to support primary AML cell survival and phenotype maintenance in cultures. Specifically, 3DON derived from osteogenically differentiated mesenchymal stem cells (MSC) from healthy and AML donors are co-cultured with primary AML cells. The AML cells under the AML_3DON niche showed enhanced viability, reduced apoptosis and maintained CD33+ CD34−phenotype, associating with elevated secretion of anti-apoptotic cytokines in the AML_3DON niche. Moreover, AML cells under the AML_3DON niche exhibited low sensitivity to two FDA-approved chemotherapeutic drugs, further suggesting the physiological resemblance of the AML_3DON niche. Most interestingly, AML cells co-cultured with the healthy_3DON niche are highly sensitive to the same sample drugs. This study demonstrates the differential responses of AML cells towards leukemic and healthy bone marrow niches, suggesting the impact of native cancer cell niche in drug screening, and the potential of re-engineering healthy bone marrow niche in AML patients as chemotherapeutic adjuvants overcoming chemoresistance, respectively.

Allogeneic hematopoietic cell transplantation for acute myeloid leukemia with BCR::ABL1 fusion.

BCR::ABL1 fusion is found in<1% of de novo acute myeloid leukemia (AML) cases and confers a poor prognosis. This Japanese nationwide survey analyzed patients with AML (n=22) and mixed phenotype acute leukemia (MPAL) (n=10) with t(9;22) or BCR::ABL1 who underwent allogeneic hematopoietic cell transplantation (allo-HCT) between 2002 and 2018. The 3-year overall survival (OS) rates were 81.3% and 56.0%, respectively (p=0.15), and leukemia-free survival (LFS) rates were 76.2% and 42.0%, respectively (p=0.10) in patients with AML and MPAL. The relapse rates were 9.5% and 14.0% (p=0.93), and the non-relapse mortality (NRM) rates were 14.3% and 44.0%, respectively (p=0.10) in patients with AML and MPAL. One in 17 patients with AML, with pre-transplant tyrosine kinase inhibitors (TKI), and three in five patients with AML, without pre-transplant TKI, did not achieve complete remission (CR) before allo-HCT (p=0.024). Among the 20 patients with known disease status after allo-HCT, 95.0% were in hematological or molecular CR. None of the four patients who received post-transplant TKI for prophylaxis or measurable residual disease relapse experienced hematological relapse. In conclusion, our results suggest that pre-transplant TKI could improve disease status before allo-HCT. Moreover, allo-HCT resulted in high OS, high LFS, low relapse, and low NRM rates in patients with AML with BCR::ABL1.

Acute Myeloid Leukemia Differentiation State and Genotype Influence Anti-Apoptotic Protein Expression, Venetoclax Sensitivity, and Survival in AML

Background: Somatic mutations (mut) and acute myeloid leukemia (AML) differentiation state (phenotype) influence sensitivity and response to treatment. However the interaction between genotype and phenotype with respect to sensitivity or resistance to venetoclax (VEN) based therapy has yet to be fully evaluated. Methods: Samples from patients (pts.) with newly diagnosed (ND) AML enrolled in Beat AML with available bulk RNA sequencing (RNAseq), whole exome sequencing, and ex vivo drug sensitivity testing (N=255) were analyzed for correlation between AML phenotype (defined using RNAseq [van Galen et. al. Cell 2019] and surface immunophenotype via multiparameter flow cytometry [MFC]), genotype, and VEN sensitivity. A monocytic score corresponding to monocytic gene expression and myeloid differentiation for each sample was included as previously defined (Bottomly et. al. Cancer Cell 2022). Clinical outcomes were assessed in an independent retrospective patient cohort (N=97) treated with frontline hypomethylating agents (HMA)+VEN at Oregon Health &amp; Science University. Categorical and continuous variables were analyzed using Fisher's or Wilcoxon rank sum testing. Time to event outcomes utilized the log-rank method with cox multivariable regression modeling. Results: Median patient (pt) age at diagnosis in the surveyed cohort was 61 years (range 20-88). AML phenotype determined using proportional deconvolution of RNAseq data was HSC/progenitor (primitive) in 31% (N=78), mixed/intermediate in 31% (N=79), cDC/monocytic (mature) in 29% (N=73), and unknown in 10% (N=25) of pts. Muts frequent in primitive vs. mature AML samples included transcription factor ( RUNX1, CEBPA, GATA2, GATA1, ETV6, IKZF1; 30% vs. 15%, p=0.050), tumor suppressor ( TP53, PHF6, WT1; 21% vs. 7%, p=0.019), and IDH1/2 (27% vs. 12%, p= 0.027). Active signaling mut ( FLT3-ITD/TKD, K/NRAS, PTPN11, BRAF) were more frequent in mature vs. primitive samples (78% vs. 44%, p &amp;lt; 0.0001), underscoring the correlation between genotype and myeloid differentiation (Fig.A). Expression of anti-apoptotic protein and myeloid differentiation markers measured using RNAseq varied by AML genotype and phenotype and corresponded to VEN resistance. The ratios of BCL2A1 (pearson R=0.62, p &amp;lt; 0.0001), CD11b (R=0.65, p &amp;lt; 0.0001), CD14 (R=0.73, p &amp;lt; 0.0001), and CLEC7A (R=0.72, p &amp;lt; 0.0001) to BCL2 positively correlated with VEN resistance (i.e., increasing area under the curve [AUC]). While IDH1/2 and NPM1 mutated samples demonstrated the highest BCL2 expression and lowest BCL2A1: BCL2 ratio and remained largely sensitive to VEN, myeloid differentiation (in particular monocytic differentiation) increased VEN resistance irrespective of genotype. For instance, classification of a primitive vs. mature phenotype further stratified ex vivo VEN sensitivity in pts with IDH1 (median AUC: 61 vs. 195, p: 0.004), IDH2 (median AUC: 61 vs. 185, p: 0.002), or NPM1 (median AUC: 60 vs. 220, p &amp;lt; 0.0001) mut (Fig.B). Overall survival (OS) following HMA+VEN was evaluated based on the presence or absence of surface immunophenotypic markers measured via MFC that correlated with myeloid differentiation. CD117+ AML (HR: 0.30 95% CI: 0.14 - 0.65, p=0.002) associated with improved OS, while inferior OS was observed with CD11b+ (HR: 2.03 95% CI: 1.07 - 3.86, p=0.031) and CD64+ AML (HR:1.86 95% CI: 0.90 - 3.85, p=0.095). When assessing the combined influence of genotype and immunophenotype on OS in CD11b+ or CD64+ AML, IDH1/2 mut (N=8) numerically improved OS compared to IDH1/2 wild-type cases (N=17) (median 12.3 vs. 5.8 months, p: 0.17). In multivariate analysis adjusted for CD11b or CD64 positivity, age, IDH1/2 mut, and secondary or therapy-related AML, CD11b or CD64 positivity (HR: 1.98 95% CI: 1.08-3.66, p=0.028) retained the strongest impact on OS. Conclusions: Genotype and phenotype both modulate VEN sensitivity in AML. Myeloid differentiation is associated with increased alternative anti-apoptotic protein expression and VEN resistance irrespective of genotype; however certain mutations (i.e., IDH1/2) may retain prognostic significance independent of myeloid differentiation. Given both factors influence VEN sensitivity, prognostic models incorporating AML phenotype and genotype may further improve risk stratification and predict response to VEN based therapy in AML.

Cohesin Haploinsufficiency Promotes Reduced Latency in Inv(16) Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a cancer of the bone marrow that affects 20,000 adults in the United States alone, with global rates increasing over the last 30 years (Heimbruch et al. 2021, Dong et al. 2020). Approximately 15% of adult AML patients harbor the chromosomal aberrations t(8;21) or inv(16), which affect the core binding complex members RUNX1 and CBFB, respectively. Together, RUNX1 and CBFB function as a transcription factor that regulates key lineage genes and proliferation in hematopoietic cells (Pulikkan and Castilla 2018). t(8;21) and inv(16) result in the expression of the fusion proteins AML1-ETO and CBFB-SMMHC, both of which have dominant negative effects on native RUNX1 as well as novel transcriptional roles (Surapally et al. 2021). Although both t(8;21) and inv(16) result in AMLs with similar characteristics, they differ in the number and type of co-occurring mutations (Opatz et al. 2020). More mutations per clone are observed in t(8;21) vs. inv(16) AMLs. Additionally, several studies have highlighted a difference between the rates of mutation of cohesin genes and t(8;21) vs. inv(16) (Faber et al. 2016, Opatz et al. 2020, Duployez et al. 2016, Jahn et al. 2020, Qin et al. 2022). Cohesin is comprised of the subunits SMC3, SMC1A, RAD21, and STAG2. The complex has a prominent role in hematopoietic cells in chromatin organization and DNA looping events that regulate gene expression and self-renewal (Heimbruch et al. 2021). Cohesin mutations are found in approximately 10% of AML cases (Ley et al 2013). Interestingly, cohesin mutations are found in 18-27% of t(8;21) AMLs, but in 0-4% of inv(16) AMLs (Faber et al. 2016, Opatz et al. 2020, Duployez et al. 2016, Jahn et al. 2020, Qin et al. 2022). The low rate of co-mutation between inv(16) and cohesin genes suggests potential synthetic lethality. However, it is also possible that cohesin mutations occur at a higher rate than reported due to biases in the age, ethnicity, or treatment status of the patients included in genome/exome sequencing studies. To test if cohesin mutations and inv(16) are synthetic lethal, we used an inv(16) knock-in mouse model ( Mx1-Cre;inv(16)) combined with a cohesin-deficient mouse ( Mx1-Cre; Smc3 fl/+) (Kuo et al. 2006, Viny et al. 2015). We followed inv(16);Smc3wt and inv(16);Smc3 -/+ animals post-Cre activation and monitored mice for leukemic development. We found that inv(16);Smc3 -/+ mice developed AML with reduced latency compared to inv(16);Smc3wt mice, suggesting cooperativity rather than synthetic lethality. To determine the mechanism by which Smc3 haploinsufficiency accelerates inv(16) leukemias, we performed ATAC sequencing and single cell RNA sequencing on hematopoietic stem and progenitor cells (HSPCs). ATAC-seq revealed a large increase in open chromatin in promoter regions in inv(16);Smc3 -/+ vs. inv(16);Smc3wt HSPCs. These open regions were enriched for binding motifs of the Ets family member Fli1. Increased expression of Fli1 was observed by western blotting in inv(16);Smc3 -/+ HSPCs. Interestingly, CBFB-SMMHC binding sites commonly contain Ets motifs (Mandoli 2014), suggesting that the increased Fli1 expression and binding site accessibility in inv(16);Smc3 -/+ may drive the observed decrease in latency. scRNA-seq revealed cluster-specific differences between inv(16);Smc3wt and inv(16);Smc3 -/+ cells. In several clusters, inv(16);Smc3 -/+ cells showed a loss of expression of genes involved in apoptosis and differentiation. Of particular interest is Gata2, as loss of Gata2 generates a more aggressive phenotype in AML (Saida et al. 2020). Therefore, Smc3 haploinsufficiency may result in Gata2 deregulation, thereby impacting disease latency. Currently, we have two potential explanations for the observed phenotype: Deregulated binding of Fli1 and its downstream transcriptional pathways and decreased expression of Gata2 in sub-populations of inv(16);Smc3 -/+ cells. Which of these mechanisms mediates the reduced latency in inv(16);Smc3 -/+ animals is an area of active investigation. Collectively, our studies show that a lack of observed co-mutation does not necessarily indicate synthetic lethality. We propose that cohesin mutations may be more common in inv(16) leukemias than has been reported and suggest that a careful consideration of age, ethnicity, and treatment status may be warranted when evaluating rates of co-mutation.

Single-Cell Proteogenomics Analysis of AML Samples Treated with Decitabine and Venetoclax Reveals Divergent Treatment Escape Mechanisms

Background: The combination therapy of hypomethylating agents and venetoclax (HMA+VEN) has emerged as a standard of care for acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. Nonetheless, significant clinical challenges persist, including relapse and treatment resistance. Previous studies have revealed genetic ( RAS/RTK pathway mutations and TP53 mutations) and phenotypic (monocytic differentiation) resistance mechanisms in the context of HMA+VEN therapy. Despite these advancements, our comprehensive understanding of the HMA+VEN resistance mechanisms remains incomplete. To gain further insights into the resistance mechanism in HMA+VEN therapy, we implemented single-cell proteogenomics analysis on longitudinally collected samples from AML patients treated with decitabine+venetoclax (DAC+VEN) trial. Methods: We performed a high-throughput, single-cell targeted DNA-antibody sequencing (DAb-seq) using Tapestri platform from Mission Bio Inc. Sixty-three AML samples sequentially collected from 33 unique patients who participated in DAC+VEN clinical trial (NCT03404193) were analyzed. Our custom DNA panel covered 37 recurrently mutated genes in AML, whereas the custom antibody panel included either 15 cell surface proteins (N = 20 patients) or 48 proteins (N = 13 patients). Cellular phenotypes were inferred based on the patterns of cell surface protein expression. Median 2,767 cells were sequenced per sample, with total 196,719 cells sequenced for this study. Results: The median age of the cohort was 70 years (range 21-84). Twenty-four (73%) patients had primary AML and 9 (27%) had secondary or therapy-related AML. Based on the FAB classification, 13 patients had M0/M1/M2, 6 had M4/M5, one had M7, and the rest had unknown subtypes. The most frequent gene mutation at baseline was DNMT3A (45%), followed by NPM1 (39%), TET2 (30%), and TP53 (24%). Among the 33 patients, 32 had evaluable treatment response; 27 (82%) were defined as responders by achieving CR or CRi, whereas 5 (15%) were non-responders. Six patients experienced relapse after responding to the therapy, and their baseline-relapse pairs were analyzed by the single-cell platform. Using the composition of cellular phenotype data, AML samples were classified into either stem-like (N = 5), progenitor-like (N = 1), predominantly monocytic (N = 4), predominantly erythroid (N = 2) and mixed population AML (mixed population of stem, progenitor, monocyte or erythroid-like population, N = 21). Genotype-phenotype analysis showed a significant correlation between the KRAS mutation and the mixed phenotype (p= 0.0403), as well as between the U2AF1 mutation and the mixed phenotype (p= 0.0471). There was no significant association between treatment response and AML phenotype. Analysis of six baseline-relapse pairs showed that relapse was associated with phenotypic shift of AML cells. Stem-like cells were largely eradicated at relapse and replaced by more differentiated cells. Among the six baseline-relapse pairs, three showed monocytic shift, one showed erythroid shift and two showed mixed monocytic/erythroid shift. Phenotypic shifts at relapse were not always associated with expansion of new genetic mutations, except for in two cases: one had monocytic shift accompanied with expansion of KRAS mutation, and another had erythroid shift accompanied with expansion of NRAS mutation. Conclusions: Phenotypic transition from stem-like cells to more differentiated cells (either monocytic or erythroid) was frequently observed in AML relapse following DAC+VEN therapy. These findings are consistent with the known dependency of these cells on non-BCL-2 anti-apoptotic proteins such as MCL-1 and BCL-xL. Close monitoring of these phenotypic alterations throughout the course of HMA+VEN treatment may potentially facilitate the precise identification of cellular populations predisposed to relapse.

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.

Perspectives and challenges of small molecule inhibitor therapy for FLT3-mutated acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous clonal disease characterized overall by an aggressive clinical course. The underlying genetic abnormalities present in leukemic cells contribute significantly to the AML phenotype. Mutations in FMS-like tyrosine kinase 3 (FLT3) are one of the most common genetic abnormalities identified in AML, and the presence of these mutations strongly influences disease presentation and negatively impacts prognosis. Since mutations in FLT3 were identified in AML, they have been recognized as a valid therapeutic target resulting in decades of research to develop effective small molecule inhibitor treatment that could improve outcome for these patients. Despite the approval of several FLT3 inhibitors over the last couple of years, the treatment of patients with FLT3-mutated AML remains challenging and many questions still need to be addressed. This review will provide an up-to-date overview of our current understanding of FLT3-mutated AML and discuss what the current status is of the available FLT3 inhibitors for the day-to-day management of this aggressive disease.

Arsenic Trioxide Combine with G-CSF Triggers Distinct TP53 Mutations Acute Myeloid Leukemia Cells Ferroptosis through TP53-SLC7A11-GPX4 Pathway

Abstract ： Background ： Acute myeloid leukemia (AML) is a complex disease with diverse molecular subtypes. Approximately 8% of AML patients carry the TP53 mutation, which is associated with an aggressive disease course and dismal survival rates. Arsenic trioxide (ATO) has been remarkably effective in treating acute promyelocytic leukemia (APL), but its clinical activity in other AML subtypes remains limited. Recent evidence indicates that ATO demonstrates potential in rescuing multiple TP53 mutations effectively, offering promise as an individualized therapeutic strategy targeting mutant TP53 in leukemic cells. Our previous study showed that the upregulation of AQP9 by G-CSF enhanced the effect of ATO in non-APL AML cells. This demonstrates that ATO holds therapeutic promise in AML cases harboring distinct TP53 mutations. Methods ： We conducted a comprehensive analysis of TP53 mutations in the Analysis of Whole Genomes cohort, encompassing patients from the TCGA and OHSU databases. The frequency and distribution of TP53 mutations were determined. Median overall survival (OS) was calculated using the Kaplan-Meier method. To functionally assess the effect of TP53 mutation on AML phenotype, various cell lines harboring distinct TP53 mutation types were used for this study, including KASUMI-1 TP53 R248Q THP-1 TP53frameshift_delHL-60 TP53 deletionMOLM13 TP53 wild-type. The cellular lipid oxidation levels were measured by a C11 BODIPY flow cytometry assay and it was also observed by fluorescence microscopy. Additionally, the RNA and protein expressions of GPX4 and SLC7A11 were analyzed by real-time quantitative Polymerase Chain Reaction (QRT-PCR) and Western Blot. Results ： In the OHSU cohort and TCGALAML database, including 1005 newly diagnosed patients with 120 TP53 mutations, we found that missense mutations are the most common TP53 mutations and R248Q has the highest mutation frequency. Furthermore, TP53 mutationwas significantly associated with lower survival ( P &amp;lt;0.001). ATO potently induced cell death in each cell line. The KASUMI-1 R248Q cell line was the most sensitive to ATO (IC50=0.3607) and showed the most pronounced change in the IC50 of ATO with the addition of ferrostatin-1(Fer-1, ferroptosis inhibitor). ATO induced cell death was prevented by the addition of lipophilic antioxidant ferrostatin-1, supporting the hypothesis that ATO induced cell death occurred by ferroptosis. In KASUMI-1 R248Q cell line, ATO increases the lipid oxidation levels (observed by C11 BODIPY flow cytometry assay and fluorescence microscopy), MDA levels and decreases the expression of GPX4 and SLC7A11 (including RNA level and protein level). Whereas, in MOLM13 TP53 wildtype cell line, ATO increases the lipid oxidation levels but cannot prevented by Fer-1. THP-1 TP53 frameshift_del and HL-60 TP53 deletion only decrease the RNA expression of GPX4 and SLC7A11, nothing else changed in ferroptosis. These suggest that ATO has a more pronounced effect on missense mutations such as TP53 R248Q. In KASUMI-1 R248Q cell line, associated with G-CSF can further promote lipid oxidation levels (observed by C11 BODIPY flow cytometry assay and fluorescence microscopy) and further reduce the expression of GPX4 and SLC7A11. These results suggest that G-CSF promotes ATO-induced ferroptosis in KASUMI-1 R248Q cell line. Conclusion: We have demonstrated that ATO induces AML with distinct TP53 mutation cell death by ferroptosis resulting from depletion of GPX4 and SLC7A11. ATO associated with G-CSF may be able to be a new therapeutic strategy for the treatment of AML with distinct TP53 mutation.

Features of the TCR repertoire associate with patients' clinical and molecular characteristics in acute myeloid leukemia.

Allogeneic hematopoietic stem cell transplant remains the most effective strategy for patients with high-risk acute myeloid leukemia (AML). Leukemia-specific neoantigens presented by the major histocompatibility complexes (MHCs) are recognized by the T cell receptors (TCR) triggering the graft-versus-leukemia effect. A unique TCR signature is generated by a complex V(D)J rearrangement process to form TCR capable of binding to the peptide-MHC. The generated TCR repertoire undergoes dynamic changes with disease progression and treatment. Here we applied two different computational tools (TRUST4 and MIXCR) to extract the TCR sequences from RNA-seq data from The Cancer Genome Atlas (TCGA) and examine the association between features of the TCR repertoire in adult patients with AML and their clinical and molecular characteristics. We found that only ~30% of identified TCR CDR3s were shared by the two computational tools. Yet, patterns of TCR associations with patients' clinical and molecular characteristics based on data obtained from either tool were similar. The numbers of unique TCR clones were highly correlated with patients' white blood cell counts, bone marrow blast percentage, and peripheral blood blast percentage. Multivariable regressions of TCRA and TCRB median normalized number of unique clones with mutational status of AML patients using TRUST4 showed significant association of TCRA or TCRB with WT1 mutations, WBC count, %BM blast, and sex (adjusted in TCRB model). We observed a correlation between TCRA/B number of unique clones and the expression of T cells inhibitory signal genes (TIGIT, LAG3, CTLA-4) and foxp3, but not IL2RA, CD69 and TNFRSF9 suggestive of exhausted T cell phenotypes in AML. Benchmarking of computational tools is needed to increase the accuracy of the identified clones. The utilization of RNA-seq data enables identification of highly abundant TCRs and correlating these clones with patients' clinical and molecular characteristics. This study further supports the value of high-resolution TCR-Seq analyses to characterize the TCR repertoire in patients.

Mutated IKZF1 is an independent marker of adverse risk in acute myeloid leukemia

Genetic lesions of IKZF1 are frequent events and well-established markers of adverse risk in acute lymphoblastic leukemia. However, their function in the pathophysiology and impact on patient outcome in acute myeloid leukemia (AML) remains elusive. In a multicenter cohort of 1606 newly diagnosed and intensively treated adult AML patients, we found IKZF1 alterations in 45 cases with a mutational hotspot at N159S. AML with mutated IKZF1 was associated with alterations in RUNX1, GATA2, KRAS, KIT, SF3B1, and ETV6, while alterations of NPM1, TET2, FLT3-ITD, and normal karyotypes were less frequent. The clinical phenotype of IKZF1-mutated AML was dominated by anemia and thrombocytopenia. In both univariable and multivariable analyses adjusting for age, de novo and secondary AML, and ELN2022 risk categories, we found mutated IKZF1 to be an independent marker of adverse risk regarding complete remission rate, event-free, relapse-free, and overall survival. The deleterious effects of mutated IKZF1 also prevailed in patients who underwent allogeneic hematopoietic stem cell transplantation (n = 519) in both univariable and multivariable models. These dismal outcomes are only partially explained by the hotspot mutation N159S. Our findings suggest a role for IKZF1 mutation status in AML risk modeling.

Cytotoxic T-lymphocytes in acute myeloid leukemia: Monitoring prognosis and guiding treatment choice.

Cytotoxic T-lymphocyte (CTL)-mediated therapy has become the central theme of cancer immunotherapy. The present study emphasized the role of CTLs in acute myeloid leukemia (AML) and aimed to understand the role of CTLs cytogenetic markers in monitoring AML prognostic outcomes and clinical treatment responses. Seurat was employed to analyze single-cell RNA sequencing data in GSE116256. CellChat was used to detect cell-cell interactions to determine the central role of CTLs. The marker genes of CTLs were extracted and randomForestSRC was employed to construct a random forest model. The prognosis, immune checkpoint expression, immune cell infiltration, immunotherapy response and drug sensitivity of AML patients were evaluated according to the model. Seven types of cellular components of AML were identified in GSE116256, and CTLs radiated the most interactions with other cell types. Random forest analysis screened out six marker genes for construction of the model. The risk score calculated according to the model was positively correlated with immune score, immune cell infiltration, expression of multiple immune checkpoints and immune effect pathway. The response rate of immunotherapy was significantly higher and more sensitive to 14 drugs in high-risk samples than in low-risk samples, whereas low-risk patients showed a higher sensitivity to six drugs. The present study emphasized the central role of CTLs in cell communication and established a random forest regression model based on its cytogenetic markers, which helps to stratify the prognosis of AML, promotes the understanding of the phenotype of AML and may also guide the treatment choice of AML patients, which contributed to stratification of AML prognosis, promoted understanding of the phenotype of AML and may guide treatment selection in patients with AML.

Evaluation of aberrant expression of CD markers in acute leukemia cells

Background and objective: Worldwide immunophenotyping by flow cytometry (FCM) in acute leukemia (AL) is the golden step in the diagnosis. It’s very common for acute leukemias to aberrantly express antigens or cluster of differentiation (CD) markers which are usually expressed in other lineages of the disease hence this study aimed at determining the prevalence of aberrancy in AL and to find out the frequency of each aberrant CD marker and their association with the clinic-hematological profile of the cases. Methods: Following history and clinical examination of enrolled patients, blood and/or bone marrow aspirate was drawn for morphological examination and immunophenotyping by FCM from 86 newly diagnosed acute leukemia cases then multiple steps procedure was applied followed by interpretation of the results. Results: The prevalence of aberrant phenotype was 46.5%. The proportional frequency of aberrant phenotype in acute myeloid leukemia (AML) was 41%, in B-acute lymphoblastic leukemia (B-ALL) was 48.8% and in T-acute lymphoblastic leukemia (T-ALL) was 66.6%. The commonest aberrant CD markers in AML were CD22 and CD2, in B-ALL were CD66c and CD13 while in T-ALL were CD13 and CD33. The aberrant phenotype harbored lower white blood cell (WBC) count and blast percentage in PB, also splenomegaly was more frequent in lymphoid positive (Ly+) AML and myeloid positive (My+) T-ALL while in B-ALL, splenomegaly was more frequent in myeloid negative (My-) B-ALL. Conclusion: Aberrant phenotype prevalence in our study sample was comparable to other studies, considerable frequency of aberrant markers is present in cases of AL and some variations exist regarding the clinical and hematological profile of the aberrant group.

Abstract A41: Single Cell Multiomic Analysis Reveals Association of TP53-mut Loss of Heterozygosity with Primitive Phenotype in Acute Myeloid Leukemia

Abstract TP53 mutations in acute myeloid leukemia (AML) are associated with copy number abnormalities (CNA), structural variants and high risk of relapse (Döhner et al., 2017; Giacomelli et al., 2018; Bernard et al. 2020). In spite of relatively high remission rates obtained by targeted therapies, TP53 mutant (TP53-mut) clones persist, invariably resulting in relapse (Short et al., 2021; Takahashi et al., 2016). Delineating the clonal architecture and the immunophenotypes of TP53-mut clones during AML therapy may provide a better understanding of the role of TP53-mutations in AML biology. Recent progress in sequencing technologies allows the integration of genotyping and phenotyping at the single cell level. Here, we took advantage of MissionBio Tapestri’s newest platform: single cell DNA + protein for simultaneous genotyping and phenotyping with 45 surface oligo-conjugated antibodies in 10 paired samples from 5 patients with TP53-mut AML before and after therapy. Samples with at least 70% viability were stained with the TotalSeq™-D Human Heme Oncology Cocktail, V1.0. Following surface marker staining, single-cell suspension, encapsulation, and barcoding were performed according to manufacturer’s instruction. For scDNA library preparation, we utilized a validated custom panel (Morita et al. 2020) consisting of 279 amplicons covering recurrent mutations in 37 genes in AML. We sequenced a total of 44,550 cells from 10 samples. We confirmed mutations reported by MD Anderson molecular diagnostic laboratory in the genes covered by the scDNA custom panel. The clonal architecture analysis distinguished between TP53-mut clones with or without loss of heterozygosity (LOH) of the normal TP53 allele. Our data show a primitive immunophenotype in TP53-mut with LOH (LOH+) clones in comparison to TP53-mut LOH- clones. We see 2.4 LOG2FC increase in CD34 and 1.8 LOG2FC increase in CD117 (p&amp;lt;0.001) in TP53-mut LOH+ clones in comparison to TP53-mut LOH- clones. Clonal evolution analysis shows that TP53-mut LOH+ clones are significantly more resistant to therapy than TP53-mut LOH-, consistent with previous publications. On the other hand, TP53-mut LOH- clones showed significantly higher levels of CD2, CD16, CD5, CD3, and CD8 among other lineage markers (LOG2FC= 1.1, 1.2, 1.4, 1.1, and 1.1 respectively; p.value &amp;lt;0.0001) compared to TP53-mut LOH+. This data indicates that TP53-mut LOH- cells can express lymphoid phenotypic markers. Single cell cytokine analysis (IsoPlexis) reveals profound lack of secreted cytokines in T-cells from TP53-mut AML. Further data from bulk-RNA sequencing, ddPCR, and CyTOF will be presented that validates a lymphoid phenotype of TP53-mut LOH-. In summary, we utilize a scDNA+protein multiomic approach to dissect clonal architecture and provide a link between genotype-phenotype in TP53-mut AML. We show that while TP53-mut LOH+ clones are exclusively primitive, TP53-mut LOH- clones retain the capacity to exist outside primitive immunophenotype and might lack differentiation block. Citation Format: Edward Ayoub, Vakul Mohanty, Yuki Nishida, Tallie Patsilevas, Mahesh Basyal, Russell Pourebrahim, Muharrem Muftuoglu, Ken Chen, Ghayas C. Issa, Michael Andreeff. Single Cell Multiomic Analysis Reveals Association of TP53-mut Loss of Heterozygosity with Primitive Phenotype in Acute Myeloid Leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A41.

Abstract 3804: Linear PVT1 isoforms and circPVT1 regulate DNA damage response and immunological pathways in acute myeloid leukemia and have a potential role in the crosstalk between leukemic cells and the tumor microenvironment

Abstract The human Plasmacytoma Variant Translocation 1 (PVT1) gene encodes for 176 linear (PVT1, lncipedia.org) and 29 circular isoforms (circPVT1, www.circbase.org). The most common isoform of circPVT1 is 410bp and is a product of back-splicing containing the whole exon 2 of PVT1 in a closed loop-like structure (hsa_circ_0001821). PVT1 and circPVT1 have oncogenic roles in several tumor types and can contribute to the suppression of anti-tumor immune responses. Our study aims to investigate whether and how PVT1 isoforms and circPVT1 promote an aggressive phenotype in acute myeloid leukemia (AML) and are involved in the crosstalk between leukemic and immune cells. Six out of the 14 PVT1 isoforms expressed by white blood cells and lymphnodes (noncode.org) were detected in a panel of AML cell lines along with circPVT1. Downregulation (KD) of groups of linear isoforms (based on sequence homology) or of circPVT1 by antisense-oligonucleotides (ASOs) in the t(8;21) KASUMI-1 and the NPM1-mut OCI-AML3 cells induced an impairment of leukemia cell growth especially when all the 6 linear isoforms were silenced by ASO combination, in both cell lines, under normoxia and hypoxia mimicking the bone-marrow microenvironment. Interestingly circPVT1-KD induced apoptosis in OCI-AML3 cells. Moreover, MYC protein expression decreased by ASO combination or circPVT1_KD in OCI-AML3 under normoxia and in KASUMI-1 under hypoxia. To further investigate the biological consequences of PVT1/circPVT1-KD, we performed RNAseq. Analysis on AML cell lines revealed that downregulation of PVT1 and, especially circPVT1, altered the expression of genes involved in RNA transport and degradation, as expected for a long non-coding genes, but also in DNA damage response (DDR) and immunological pathways, as also confirmed by transcriptomic data of primary AML cases and proteomic analysis of both cell lines. Based on these novel findings, we tested the downregulation of PVT1/circPVT1 in combination with inhibition of ATM or ATR, key DDR transducers. Interestingly, circPVT1-KD sensitized OCI-AML3 to ATM (71% vs 95% live cells) or ATR (75% vs 90% live cells) inhibitors. PVT1 and circPVT1 expression was also detected at the level of cell-free RNA in primary AML samples, the latter showing higher levels. We then analyzed extracellular vesicles (EVs) from AML peripheral blood and healthy subjects. EVs from patients were larger, increased in number and expressed higher levels of myeloid lineage markers. Finally AML EVs had more circPVT1 cargo. In conclusion, we uncovered novel potential roles of PVT1 isoforms and circPVT1 in the DDR and in the tumor microenvironment that pave the way for novel therapeutic combinations. Citation Format: Martina Ghetti, Lorenzo Ledda, Ivan Vannini, Eugenio Fonzi, Maria Teresa Bochicchio, Tristan Cardon, Andrea Ghelli Luserna di Rorà, Matteo Paganelli, Chiara Servili, Francesco Fabbri, Sabina Marianini, Giovanni Marconi, Michela Rondoni, Roberta Chicchi, Rino Biguzzi, Francesco Lanza, Michel Salzet, Giovanni Martinelli, Giorgia Simonetti. Linear PVT1 isoforms and circPVT1 regulate DNA damage response and immunological pathways in acute myeloid leukemia and have a potential role in the crosstalk between leukemic cells and the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3804.

Characterization of cuproptosis identified immune microenvironment and prognosis in acute myeloid leukemia.

Recent studies have reported that cuproptosis, a novel cell death pathway, strongly correlates with mitochondrial metabolism. In addition, the studies reported that cuproptosis plays a role in the development of several cancers and is regulated by protein lipoylation. During cuproptosis, copper binds to the lipoylated proteins and mediates cancer progression. However, the role of cuproptosis in acute myeloid leukemia (AML) patients is yet to be explored. This study curated seven cuproptosis-related-genes (CRGs): FDX1, DLAT, PDHB, PDHA1, DLD, LIAS, and LIPT1 to determine cuproptosis modification patterns and the CRGs signature in AML. The CIBERSORT and ssGSEA algorithms were utilized to evaluate the infiltration levels of different immune cell subtypes. A cuproptosis score system based on differentially expressed genes (DEGs) was constructed using the least absolute shrinkage and selection operator (LASSO) regression analysis. The developed cuproptosis score system was validated using two immunotherapy datasets, IMvigor210 and GSE78220. Three distinct cuproptosis regulation patterns were identified using the Beat AML cohort. The results demonstrated that the three cuproptosis regulation patterns were correlated with various biological pathways and clinical outcomes. Tumor microenvironment (TME) characterization revealed that the identified cuproptosis regulation patterns were consistent with three immune profiles: immune-desert, immune-inflamed, and immune-excluded. The AML patients were grouped into low- and high-score groups based on the cuproptosis score system abstracted from 486 cuproptosis-related DEGs. Patients with lower cuproptosis scores were characterized by longer survival time and attenuated immune infiltration. It was found that lower cuproptosis scores were strongly correlated with lower somatic mutation frequency. Moreover, patients with lower cuproptosis scores presented more favorable immune responses and dual clinical benefits among external validation cohorts. Cuproptosis phenotypes are significantly correlated with immune microenvironment complexity and variety. Cuprotopsis regulates the response of cancer cells to the immune system. Quantitatively assessing cuproptosis phenotypes in AML improves the understanding and knowledge regarding immune microenvironment characteristics and promotes the development of therapeutic interventions.

Long Noncoding RNA Expression Independently Predicts Outcome in Pediatric Acute Myeloid Leukemia.

Optimized strategies for risk classification are essential to tailor therapy for patients with biologically distinctive disease. Risk classification in pediatric acute myeloid leukemia (pAML) relies on detection of translocations and gene mutations. Long noncoding RNA (lncRNA) transcripts have been shown to associate with and mediate malignant phenotypes in acute myeloid leukemia (AML) but have not been comprehensively evaluated in pAML. To identify lncRNA transcripts associated with outcomes, we evaluated the annotated lncRNA landscape by transcript sequencing of 1,298 pediatric and 96 adult AML specimens. Upregulated lncRNAs identified in the pAML training set were used to establish a regularized Cox regression model of event-free survival (EFS), yielding a 37 lncRNA signature (lncScore). Discretized lncScores were correlated with initial and postinduction treatment outcomes using Cox proportional hazards models in validation sets. Predictive model performance was compared with standard stratification methods by concordance analysis. Training set cases with positive lncScores had 5-year EFS and overall survival rates of 26.7% and 42.7%, respectively, compared with 56.9% and 76.3% with negative lncScores (hazard ratio, 2.48 and 3.16; P < .001). Pediatric validation cohorts and an adult AML group yielded comparable results in magnitude and significance. lncScore remained independently prognostic in multivariable models, including key factors used in preinduction and postinduction risk stratification. Subgroup analysis suggested that lncScores provide additional outcome information in heterogeneous subgroups currently classified as indeterminate risk. Concordance analysis showed that lncScore adds to overall classification accuracy with at least comparable predictive performance to current stratification methods that rely on multiple assays. Inclusion of the lncScore enhances predictive power of traditional cytogenetic and mutation-defined stratification in pAML with potential, as a single assay, to replace these complex stratification schemes with comparable predictive accuracy.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The 8p11 myeloproliferative syndrome: Genotypic and phenotypic classification and targeted therapy

EMS(8p11 myeloproliferative syndrome, EMS) is an aggressive hematological neoplasm with/without eosinophilia caused by a rearrangement of the FGFR1 gene at 8p11-12. It was found that all cases carry chromosome abnormalities at the molecular level, not only the previously reported chromosome translocation and insertion but also a chromosome inversion. These abnormalities produced 17 FGFR1 fusion genes, of which the most common partner genes are ZNF198 on 13q11-12 and BCR of 22q11.2. The clinical manifestations can develop into AML (acute myeloid leukemia), T-LBL (T-cell lymphoblastic lymphoma), CML (chronic myeloid leukemia), CMML (chronic monomyelocytic leukemia), or mixed phenotype acute leukemia (MPAL). Most patients are resistant to traditional chemotherapy, and a minority of patients achieve long-term clinical remission after stem cell transplantation. Recently, the therapeutic effect of targeted tyrosine kinase inhibitors (such as pemigatinib and infigratinib) in 8p11 has been confirmed in vitro and clinical trials. The TKIs may become an 8p11 treatment option as an alternative to hematopoietic stem cell transplantation, which is worthy of further study.

Graphdiyne oxide nanosheets display selective anti-leukemia efficacy against DNMT3A-mutant AML cells.

DNA methyltransferase 3 A (DNMT3A) is the most frequently mutated gene in acute myeloid leukemia (AML). Although chemotherapy agents have improved outcomes for DNMT3A-mutant AML patients, there is still no targeted therapy highlighting the need for further study of how DNMT3A mutations affect AML phenotype. Here, we demonstrate that cell adhesion-related genes are predominantly enriched in DNMT3A-mutant AML cells and identify that graphdiyne oxide (GDYO) display an anti-leukemia effect specifically against these mutated cells. Mechanistically, GDYO directly interacts with integrin β2 (ITGB2) and c-type mannose receptor (MRC2), which facilitate the attachment and cellular uptake of GDYO. Furthermore, GDYO binds to actin and prevents actin polymerization, thus disrupting the actin cytoskeleton and eventually leading to cell apoptosis. Finally, we validate the in vivo safety and therapeutic potential of GDYO against DNMT3A-mutant AML cells. Collectively, these findings demonstrate that GDYO is an efficient and specific drug candidate against DNMT3A-mutant AML.