AML Samples Research Articles

Glutamine metabolism-related genes predict the prognostic risk of acute myeloid leukemia and stratify patients by subtype analysis

BackgroundAcute myeloid leukemia (AML) is a genetically heterogeneous disease in which glutamine (Gln) contributes to AML progression. Therefore, this study aimed to identify potential prognostic biomarkers for AML based on Gln metabolism-related genes.MethodsGln-related genes that were differentially expressed between Cancer Genome Atlas-based AML and normal samples were analyzed using the limma package. Univariate, least absolute shrinkage, selection operators, and stepwise Cox regression analyses were used to identify prognostic signatures. Risk score-based prognostic and nomogram models were constructed to predict the prognostic risk of AML. Subsequently, consistent cluster analysis was performed to stratify patients into different subtypes, and subtype-related module genes were screened using weighted gene co-expression network analysis.ResultsThrough a series of regression analyses, HGF, ANGPTL3, MB, F2, CALR, EIF4EBP1, EPHX1, and PDHA1 were identified as potential prognostic biomarkers of AML. Prognostic and nomogram models constructed based on these genes could significantly differentiate between high- and low-risk AML with high predictive accuracy. The eight-signature also stratified patients with AML into two subtypes, among which Cluster 2 was prone to a high risk of AML prognosis. These two clusters exhibited different immune profiles. Of the subtype-related module genes, the HOXA and HOXB family genes may be genetic features of AML subtypes.ConclusionEight Gln metabolism-related genes were identified as potential biomarkers of AML to predict prognostic risk. The molecular subtypes clustered by these genes enabled prognostic risk stratification.

Lipopolymer/siRNA complexes engineered for optimal molecular and functional response with chemotherapy in FLT3-mutated acute myeloid leukemia

Approximately 25% of newly diagnosed AML patients display an internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene. Although both multi-targeted and FLT3 specific tyrosine kinase inhibitors (TKIs) are being utilized for clinical therapy, drug resistance, short remission periods, and high relapse rates are challenges that still need to be tackled. RNA interference (RNAi), mediated by short interfering RNA (siRNA), presents a mechanistically distinct therapeutic platform with the potential of personalization due to its gene sequence-driven mechanism of action. This study explored the use of a non-viral approach for delivery of FLT3 siRNA (siFLT3) in FLT3-ITD positive AML cell lines and primary cells as well as the feasibility of combining this treatment with drugs currently used in the clinic. Treatment of AML cell lines with FLT3 siRNA nanocomplexes resulted in prominent reduction in cell proliferation rates and induction of apoptosis. Quantitative analysis of relative mRNA transcript levels revealed downregulation of the FLT3 gene, which was accompanied by a similar decline in FLT3 protein levels. Moreover, an impact on leukemic stem cells was observed in a small pool of primary AML samples through significantly reduced colony numbers. An absence of a molecular response post-treatment with lipopolymer/siFLT3 complexes in peripheral blood mononuclear cells, obtained from healthy individuals, denoted a passive selectivity of the complexes towards malignant cells. The effect of combining lipopolymer/siFLT3 complexes with daunorubucin and FLT3 targeting TKI gilteritinib led to a significant augmentation of anti-leukemic activity. These findings demonstrate the promising potential of RNAi implemented with lipopolymer complexes for AML molecular therapy. The study prospectively supports the addition of RNAi therapy to current treatment modalities available to target the heterogeneity prevalent in AML. Statement of significanceWe show that a clinically validated target, the FLT3 gene, can be eradicated in leukemia cells using non-viral RNAi. We validated these lipopolymers as effective vehicles to deliver nucleic acids to leukemic cells. The potency of the lipopolymers was superior to that of the ‘gold-standard’ delivery agent, lipid nanoparticles (LNPs), which are not effective in leukemia cells at clinically relevant doses. Mechanistic studies were undertaken to probe structure-function relationships for effective biomaterial formulations. Cellular and molecular responses to siRNA treatment have been characterized in cell models, including leukemia patient-derived cells. The use of the siRNA therapy with clinically used chemotherapy was demonstrated.

Rational combinatorial targeting by adapter CAR-T-cells (AdCAR-T) prevents antigen escape in acute myeloid leukemia

Targeting AML by chimeric antigen receptor T-cells (CAR-T) is challenging due to the promiscuous expression of AML-associated antigens in healthy hematopoiesis and high degree of inter- and intratumoral heterogeneity. Here, we present single-cell expression data of AML-associated antigens in 30 primary pediatric AML samples. We identified CD33, CD38, CD371, IL1RAP and CD123 as the most frequently expressed. Notably, high variability was observed not only across the different patient samples but also among leukemic cells of the same patient suggesting the necessity of multiplexed targeting approaches. To address this need, we utilized our modular Adapter CAR (AdCAR) platform, enabling precise qualitative and quantitative control over CAR-T-cell function. We show highly efficient and target-specific activity for newly generated adapter molecules (AMs) against CD33, CD38, CD123, CD135 and CD371, both in vitro and in vivo. We reveal that inherent intratumoral heterogeneity in antigen expression translates into antigen escape and therapy failure to monotargeted CAR-T therapy. Further, we demonstrate in PDX models that rational combinatorial targeting by AdCAR-T-cells can cure heterogenic disease. In conclusion, we elucidate the clinical relevance of heterogeneity in antigen expression in pediatric AML and present a novel concept for precision immunotherapy by combinatorial targeting utilizing the AdCAR platform.

Overexpression of RBM4 promotes acute myeloid leukemia cell differentiation by regulating alternative splicing of TFEB

Alternative splicing (AS) is an efficient and ubiquitous transcriptional regulatory mechanism that expands the coding capacity of the genome and is associated with the occurrence and progression of cancer. The differentiation-promoting regimen is a potential therapeutic approach in cancer treatment. In this study, we screened NPMc-positive and NPMc-negative AML samples from the Cancer Genome Atlas (TCGA), focusing on the splicing factor RBM4 and its splicing mechanism on the target gene TFEB, which are most relevant to the prognosis of AML. We also investigated the impact of the TFEB-dominant spliceosome on autophagy and differentiation of THP-1 and K562 cells. The results showed that RBM4 recognized the CU-rich sequence in intron 8 of TFEB, increasing the production of the TFEB-L spliceosome, which promoted autophagy. Overexpression of RBM4 increased autophagy and promoted cell differentiation. The combination of TFEB-L with the therapeutic drug rapamycin further promoted the differentiation of leukemia cell lines and primary leukemia cells in AML patients. This study suggested that overexpression of RBM4 could promote cell differentiation by promoting the production of the TFEB-dominant spliceosome, demonstrating the potential of the TFEB-dominant spliceosome combined with chemotherapy drugs to promote leukemia cell differentiation and improve patient prognosis.

Abstract A012: Targeting proteasome vulnerabilities for the treatment of monosomy 7 associated blood disorders

Abstract Monosomy 7 is among the most frequent cytogenetic abnormalities in hematological disorders and is associated with short survival and drug resistance. Despite its high prevalence and detrimental impact, the therapeutic vulnerabilities underlying monosomy 7-associated blood disorders remain largely elusive, impeding progress toward improved patient care. The homeostatic cellular requirement for a normal dosage of essential genes creates an opportunity to target vulnerabilities that arise due to reduced levels of proteins encoded by a haploinsufficient essential gene. Briefly, the loss of one copy of a dosage-sensitive essential gene (gene X), in combination with the inhibition of itself or a related gene (gene Y), or an associated pathway results in lethal consequences for cells. The remarkable and selective clinical efficacy of lenalidomide for the treatment of del(5q) MDS has demonstrated how allelic haploinsufficiency underlies the sensitivity to this drug by synthetic lethality. Differential expression analysis of gene and protein expression in primary AML samples with monosomy 7 revealed significant downregulation of multiple proteasome pathway members at the protein level, but not at the RNA level. Primary AML samples with -7/del(7q) exhibited increased sensitivity (low IC50) to the proteasome inhibitor bortezomib, as evidenced by two independent ex vivo drug screening cohorts (the Beat AML and the FIMM study). Chromosome 7 harbors four proteasome subunits, PSMA2, PSMC2, PSMG3, and SEM1. We performed gene expression, protein expression, copy number analysis, and individual gene knockout experiments. The results have revealed PSMA2 to be a haploinsufficient essential gene on chromosome 7. PSMA2 knockout confers leukemia a growth disadvantage for multiple AML cell clines in both TP53 wild-type and knockout backgrounds. We generated PSMA2 isogenic hemizygous deletion and diploid single-cell clones. PSMA2 hemizygous deletion cells exhibited approximately half the protein expression compared to diploid controls, confirming that PSMA2 is a haploinsufficient gene. PSMA2hemizygous deletion single-cell clones showed significantly enhanced sensitivity to all three evaluated proteasome inhibitors (bortezomib, ixazomib, and carfilzomib), aligning with the sensitivity observed in primary -7/del(7q) leukemia samples. PSMA2 hemizygous deletion cell clones displayed increased p38 and decreased pERK levels upon treatment with proteasome inhibitors, potentially contributing to their increased sensitivity to proteasome inhibitors. Proteomics analysis and in vivo validation is ongoing. As such, we have identified haploinsufficient essential gene PSMA2 mediated proteasome pathway vulnerability in monosomy 7 associated leukemia and further showed that proteasome inhibitors as promising therapeutic approaches for treating hematological disorders associated with monosomy 7. Citation Format: Haijiao Zhang, Basil Allen, Daniel Bottomly, Peter Ryabinin, Schannon K. Targeting proteasome vulnerabilities for the treatment of monosomy 7 associated blood disorders [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A012.

Abstract 7419: Seq2Karyotype (S2K): A method for deconvoluting heterogeneity of copy number alterations using single-sample whole-genome sequencing data

Abstract Broad copy number alterations (CNAs) at chromosomal band resolution have been used as cancer diagnostic and prognostic biomarkers for decades. These events have been characterized by cytogenetic imaging, an approach which is powerful for assessing heterogeneity but limited for locus fine mapping. While CNA detection by next-generation sequencing has become a standard analysis, existing methods rarely model CNA heterogeneity in bulk tumor samples and often require a paired normal sample for control. To overcome these limitations, we developed Seq2Karyotype (S2K, https://github.com/chenlab-sj/Seq2Karyotype), a new algorithm for in-silico karyotyping using single-sample whole-genome sequencing (WGS) data. S2K performs joint modeling of read-depth and allelic imbalance (AI) of high-quality heterozygous SNPs to identify reference diploid regions, followed by modeling and segmenting the deviation from the reference. Empirical coverage and AI of segmented regions are fitted to models of single and admixed CNAs to estimate clonality. The final karyotyping considers both the model fitness and minimization of evolution steps.To evaluate S2K’s performance, we analyzed two cell lines commonly used for benchmark test: COLO829 (melanoma) and HCT1395 (breast cancer); both had single-cell (sc) WGS for validation. For COLO829, S2K replicated the four populations detected by scWGS but derived a different clonality estimate. The predominant clone, defined by loss of 1p, 10p, and chr18, was estimated to have 67% cellular fraction (CF) by bulk sample analysis of S2K in contrast to the 10% CF by scWGS analysis. In HCT1395, S2K identified three new CNAs present in >50% of the cells of the matching germline sample which were subsequently validated by FISH, karyotyping and SKY mapping.To demonstrate S2K’s utility, we analyzed three diverse data sets: 17 neuroblastoma cell lines, 24 pediatric AML samples with karyotyping data, and two blood samples from children with myelodysplastic syndromes (MDS) known to harbor mosaic uniparental disomy (UPD). CNA-based intra-tumor heterogeneity was detected in 88% (15/17) of the neuroblastoma cell lines, comprised of 2-4 distinct populations with diverse ranges of CF (~10%-90%) and varying CNA patterns (e.g. admixture of tetraploid and diploid cells), which were validated by cytogenetics or scWGS. In the patient AML samples, S2K detected >95% of the previously reported cytogenetic events and 30% of additional copy-neutral loss-of-heterozygosity events. The mosaic UPD events in MDS patients were detected with projected clonality of 60% and 25%, respectively. These results not only demonstrate the accuracy of in-silico karyotyping performed by S2K but also reveal the dynamic intra-tumor heterogeneity in cancer cell lines, which may impact the design and interpretation of future experiments using these cell lines. Citation Format: Limeng Pu, Karol Szlachta, Virginia Valentine, Xiaolong Chen, Jian Wang, Dennis Kennetz, Daniel Putnam, Sivaraman Natarajan, Li Dong, Thomas Look, Marcin Wlodarski, Lu Wang, Steven Burden, John Easton, Xiang Chen, Jinghui Zhang. Seq2Karyotype (S2K): A method for deconvoluting heterogeneity of copy number alterations using single-sample whole-genome sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7419.

Abstract 1646: IL-23R is a regulator of the mitotic spindle and is critical for cell viability in AML

Abstract To identify novel biological vulnerabilities in AML, we searched for gene ontologies that were upregulated in AML compared to normal hematopoietic cells. Of the upregulated processes, the mitotic spindle ontology was a top hit. Unexpectedly, expression of the IL-23 receptor (IL-23R) showed a strong positive correlation with the mitotic spindle ontology. IL-23R is a cell surface cytokine receptor that is canonically expressed on T cells, but not known to have a role in AML. In 7 of 7 tested AML cell lines and 15 of 20 primary AML patient samples, IL-23R protein was expressed >2 fold higher compared to mean IL-23R in normal mononuclear hematopoietic cells (n=5) and CD34+ cells (n=3). IL-23R is classically a T cell surface receptor and we confirmed localization to the cell surface in T cells. However, only small amounts of IL-23R were present on the cell surface of AML cell lines and primary AML samples. Rather, in AML cells, primary AML samples, and AML stem cells, IL-23R was located intracellularly as determined by 4 different modalities and 4 different antibodies directed against 4 different epitopes of the receptor. Of note, only the full-length IL-23R protein was detected by immunoblotting. We also detected IL-23R’s heterodimeric partner, IL12Rβ1, in AML cells. To understand the function of intracellular IL-23R, we used BioID mass spectrometry to identify proteins that interacted with IL-23R. Pathway analysis of interacting proteins identified mitotic spindle formation as the top pathway. Using Proximity Ligation Assays, we validated interactions with mitotic spindle proteins, and BioID hits, NUMA, TMEM201, TACC1, and BAG6 in AML cells. By confocal microscopy, we demonstrated that IL-23R colocalized with the mitotic spindle and centrosomes in AML cells and primary AML samples. IL-23R interacted with the mitotic spindle via its (S/T)x(I/L)P motif (amino acids 588-591). IL-12Rβ1 was also found in complex with IL-23R at the mitotic spindle in AML cells. Additionally, it was discovered that the cytokine, IL-23, promoted translocation of IL-23R to the mitotic spindle through clathrin-mediated endocytosis. Knockdown and knockout of IL-23R in AML cells led to dysregulation of the mitotic spindle with multipolarity, lagging chromosomes, and other spindle defects. Knockdown of IL-23R also reduced proliferation, clonogenic growth, and marrow engraftment of AML cells and primary AML samples. In contrast, knockdown of IL-23R in normal CD34+ cells did not impair engraftment. Likewise, constitutive homozygote IL-23R knockout mice had no difference in complete blood counts and stem cell number/function compared to wild type. In summary, IL-23R interacts with the mitotic spindle and centrosome where it regulates mitotic spindle formation and is critical for AML cell viability. Thus, we discover a novel function for IL-23R and a potential therapeutic target for this disease. Citation Format: Nathan Duong, Dilshad H. Khan, Geethu E. Thomas, Rose Hurren, Jong Bok Lee, Jonathan St-Germain, Lily Drimmer, Yongran Yan, Neil Maclean, Marcela Gronda, Vincent Rondeau, Brandon D. Brown, Courtney L. Jones, Hong Chang, Andrea Arruda, Mark D. Minden, Li Zhang, Steven M. Kornblau, Brian Raught, Vito Spadavecchio, Aaron D. Schimmer. IL-23R is a regulator of the mitotic spindle and is critical for cell viability in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1646.

Abstract 5898: Inhibition of the LONP1 ATPase domain induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells

Abstract Compared to normal hematopoietic cells, AML cells exhibit a heightened reliance on mitochondrial metabolism for survival and proliferation. To support this unique phenotype, AML cells increase import of nuclear-encoded mitochondrial proteins which must be properly folded by mitochondrial chaperones, proteases, and heat shock proteins. Failure to properly fold these precursors leads to protein aggregation, mitochondrial dysfunction, and cell death. To evaluate the reliance of AML cells on maintaining mitochondrial proteostasis, we assessed gene dependency datasets (eg: depmap.org) and identified the mitochondrial protease LONP1, as the top essential gene. LONP1 is a matrix-localized nuclear-encoded AAA+ protease. Proteins are unfolded by its ATPase domain and degraded by its serine-catalyzed proteolytic domain. Compared to hematopoietic cells, LONP1 mRNA was overexpressed in AML across three publicly available datasets. By immunoblotting, LONP1 protein was increased in 16/30 primary AML samples compared to bulk (n=8) and CD34+ blood cells (n=3). LONP1 genetic depletion reduced the growth and viability of OCI-AML2, OCI-M2, NB4, and TEX leukemia cells as well as primary AML cells. LONP1 depletion increased aggregated mitochondrial proteins and reactive oxygen species (ROS) production while reducing mitochondrial respiration and membrane potential. To determine the domain of LONP1 that was necessary for mitochondrial protein solubility, mitochondrial function, and AML survival, we over-expressed wild type, ATPase dead (E591A), or proteolytically dead (S855A) LONP1 cDNA in OCI-AML2 cells and knocked down endogenous LONP1 with shRNA targeting the 3’UTR of the endogenous gene. Wild type or proteolytically dead (S855A) but not the ATPase mutant (E591A) rescued mitochondrial protein solubility, mitochondrial respiration, ROS generation, membrane potential, and cell viability. Thus, the ATPase domain but not the proteolytic activity of LONP1 is necessary for mitochondrial proteostasis. Bardoxolone methyl (CDDO-Me) is a synthetic triterpenoid that allosterically inhibits the LONP1 ATPase site. CDDO-Me killed OCI-AML2 and NB4 cells with IC50 values of 178.5±29.7 and 156.5±39.7 nM, respectively. CDDO-Me (200 nM) killed 3 out of 4 primary AML samples with high LONP1 expression but none of the 10 primary AML samples with low LONP1. Likewise, CDDO-Me induced mitochondrial protein aggregation in AML cell lines and primary AML patient samples with high LONP1 expression but not AML cells or primary AML patient samples with low LONP1. In summary, LONP1 is over-expressed in a subset of AML cells and primary samples where it maintains mitochondrial protein solubility. Selective inhibition of the LONP1 ATPase domain leads to mitochondrial protein aggregation and AML cell death, representing a novel therapeutic strategy for AML. Citation Format: Matthew Tcheng, Marcela Gronda, Rose Hurren, Lan Xin Zhang, Chaitra Sarathy, Yongran Yan, Andrea Arruda, Mark D. Minden, Aaron D. Schimmer. Inhibition of the LONP1 ATPase domain induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5898.

Abstract 5239: Discovery of AML associated therapeutic targets

Abstract Chimeric Antigen Receptor T-cell (CART) immunotherapy has shown significant success in the treatment of hematological malignancies. However, their efficacy in AML is limited due to the disease's genetic diversity and lack of uniform antigen targets. To identify potential AML cell-surface markers, we employed a comprehensive approach that involved the utilization of 3' single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing (RNA-seq), and enhanced whole exome sequencing (eWES). A total of 21 samples were analyzed, comprising 18 distinct AML patients and 3 healthy bone marrow aspirates, serving as controls. We conducted scRNAseq on 21 samples, yielding a total of 185,106 high-quality cells, which were effectively clustered into 38 unbiased groups. Notably, tumor clusters exhibited highest copy number variations and formed separate clusters from other non-tumor immune cells. To uncover key functional pathways, we conducted pathway enrichment analysis comparing tumor cells to non-tumor cells. Our findings demonstrated a significant overrepresentation of cellular processes related to Allograft rejection, IL2/STAT5 signaling, IFN-gamma response and apoptosis. To identify AML specific markers, we performed an unbiased systematic search of the scRNAseq dataset for genes with unique expression patterns in tumor cells compared to the healthy bone marrow controls. Our analysis identified differentially expressed genes, with many genes displaying relative cell and tissue specificity. Notably, among these genes, we unveiled key potential targets with cell surface specificity, including novel markers alongside previously known ones. Noteworthy candidates, such as ADGRE2, CD33, CD123, CLEC12A, and FLT3 and understudied novel markers such as CD86, CSF1R, S100 proteins and VMP1, were prominently expressed, showcasing their potential significance as targets for engineered immunotherapy. To validate and consolidate our discoveries, we conducted a cross-referencing exercise by comparing our scRNAseq data against bulk RNA data from primary human AML samples and cell lines. Bulk global proteomics, flow cytometry-based and CART generation studies are ongoing to confirm our discoveries. Our rigorous computational and experimental approach provides valuable insights into tumor-specific cell surface proteins, which hold promise as potential targets for innovative and precision immunotherapeutic interventions in the realm of AML treatment. Citation Format: Omar M. Ibrahim, Michael P. Rettig, Reyka G. Jayasinghe, Li Ding, John F. DiPersio. Discovery of AML associated therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5239.

Single-Cell Transcriptome Analysis of Acute Myeloid Leukemia Cells Using Methanol Fixation and Cryopreservation.

The application of single-cell RNA sequencing has greatly improved our understanding of various cellular and molecular mechanisms involved in physiological and pathophysiological processes. However, obtaining living cells for this technique can be difficult under certain conditions. To solve this problem, the methanol fixation method appeared as a promising alternative for routine clinical use. In this study, we selected two AML samples that had been fixed in methanol for 12-18 months. Once the cells were rehydrated, these samples were subjected to single-cell RNA sequencing. We then compared the results obtained from these samples with those obtained from the same samples cryopreserved in DMSO. We used a previously validated methanol fixation protocol to perform scRNA-seq on DMSO cryopreserved cells and cells fixed in methanol for more than one year. Preliminary results show that methanol fixation induces some genetic and transcriptional modification compared with DMSO cryopreservation but remains a valuable method for single-cell analysis of primary human leukemia cells. The initial findings from this study highlight certain resemblances in methanol fixation over a 12-month period and cryopreservation with DMSO, along with associated transcriptional level modifications. However, we observed genetic degradation in the fixation condition when extending beyond one year. Despite certain study limitations, it is evident that short-term methanol fixation can be effectively used for leukemia blast samples. Its ease of implementation holds the potential to simplify the integration of this technique into routine clinical practice.

Epigenetic signature of ionizing radiation in therapy-related AML patients

Therapy-related acute myeloid leukaemia (t-AML) is a late side effect of previous chemotherapy (ct-AML) and/or radiotherapy (rt-AML) or immunosuppressive treatment. t-AMLs, which account for ∼10–20 % of all AML cases, are extremely aggressive and have a poor prognosis compared to de novo AML. Our hypothesis is that exposure to radiation causes genome-wide epigenetic changes in rt-AML. An epigenome-wide association study was undertaken, measuring over 850K methylation sites across the genome from fifteen donors (five healthy, five de novo, and five t-AMLs). The study predominantly focussed on 94K sites that lie in CpG-rich gene promoter regions. Genome-wide hypomethylation was discovered in AML, primarily in intergenic regions. Additionally, genes specific to AML were identified with promoter hypermethylation. A two-step validation was conducted, both internally, using pyrosequencing to measure methylation levels in specific regions across fifteen primary samples, and externally, with an additional eight AML samples. We demonstrated that the MEST and GATA5 gene promoters, which were previously identified as tumour suppressors, were noticeably hypermethylated in rt-AML, as opposed to other subtypes of AML and control samples. These may indicate the epigenetic involvement in the development of rt-AML at the molecular level and could serve as potential targets for drug therapy in rt-AML.

Abstract B122: 2 for 1: Targeting tumor-associated macrophages and cancer cells with a novel MERTK-targeting antibody-drug conjugate (ADC)

Abstract MERTK, a member of the TAM family receptor tyrosine kinases, is overexpressed in various solid and hematologic cancers. MERTK signaling drives multiple processes such as proliferation, survival, invasion and drug resistance. Furthermore, MERTK is expressed on M2 tumor-associated macrophages (TAMs), playing a role in the production of immunosuppressive cytokines and efferocytosis. Within normal tissue, MERTK is also expressed in the retinal pigment epithelium (RPE). Our RNA-DRIVErTM platform identified MERTK as a key driver of metastatic progression and thus a potentially potent target for therapeutic intervention. Our MERTK-targeting humanized monoclonal antibody, RGX-019, is internalized by both MERTK-expressing cancer cells and macrophages. In M2 macrophages, RGX-019 induced expression of pro-inflammatory M1 macrophage type cytokines. To effectively target MERTK in both cancer cells and immune-suppressive macrophages, we developed an MMAE antibody drug conjugate (ADC). Here, we assessed the anti-tumor efficacy and retinal safety of RGX-019-MMAE and examined MERTK expression in both solid and hematologic cancers. RGX-019-MMAE induced cytotoxicity against MERTK-expressing cancer cells and mediated bystander anti-tumor efficacy in vitro. In xenograft models, RGX-019-MMAE demonstrated robust anti-tumor efficacy, including complete tumor regressions in various cancers such as TNBC, melanoma, and multiple myeloma. Anti-tumor efficacy of RGX-019-MMAE was associated with improved overall survival. RGX-019-MMAE suppressed syngeneic tumor growth in a mouse model expressing human MERTK extracellular domain, and lacked anti-tumor efficacy in animals not expressing human MERTK. Anti-tumor efficacy was associated with reduced M2 macrophage levels in tumors. Given that RGX-019 only binds human but not murine MERTK, these findings suggest that RGX-019-MMAE targets the host tumor microenvironment to mediate anti-tumor efficacy. Immunohistochemical analysis revealed broad MERTK expression in multiple human cancers with highest expression detected in ovarian, NSCLC, and urothelial cancers. In primary AML samples, significantly higher expression was detected in M5 subtype samples, those with PTPN11 mutations or t(9;11) translocations. Higher MERTK expression in AML correlated with significantly worse patient survival. MERTK in the RPE is involved in maintenance of retinal homeostasis. After a 28-day treatment with RGX-019-MMAE, we observed no signs of retinal degeneration in a transgenic mouse expressing human MERTK extracellular domain. The lack of retinal toxicity can be explained by the partial agonist activity of RGX-019 and absence of MMAE cytotoxicity against non-proliferating RPE cells. The substantial anti-tumor efficacy of RGX-019-MMAE in multiple aggressive tumors, along with the limited expression in normal tissues, reveal MERTK as a promising novel target for an ADC approach. Our findings support the continued advancement of RGX-019-MMAE for the treatment of solid and hematologic cancers through targeting of both TAMs and cancer cells. Citation Format: Shugaku Takeda, PuiChi Lo, Daniel Schefer, Maryam Siddiqui, Anudishi Tyagi, Venkata Lokesh Battula, Abhishek Maiti, Sohail Tavazoie, Masoud Tavazoie, David Martin Darst, Isabel Kurth. 2 for 1: Targeting tumor-associated macrophages and cancer cells with a novel MERTK-targeting antibody-drug conjugate (ADC) [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 B122.

GNF-7, a novel FLT3 inhibitor, overcomes drug resistance for the treatment of FLT3‑ITD acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation accounts for a large proportion of AML patients and diagnosed with poor prognosis. Although the prognosis of FLT3-ITD AML has been greatly improved, the drug resistance frequently occurred in the treatment of FLT3 targeting drugs. GNF-7, a multitargeted kinase inhibitor, which provided a novel therapeutic strategy for overriding leukemia. In this study, we explored the antitumor activity of GNF-7 against FLT3-ITD and clinically-relevant drug resistance in FLT3 mutant AML.MethodsGrowth inhibitory assays were performed in AML cell lines and Ba/F3 cells expressing various FLT3 mutants to evaluate the antitumor activity of GNF-7 in vitro. Western blotting was used to examine the inhibitory effect of GNF-7 on FLT3 and its downstream pathways. Molecular docking and cellular thermal shift assay (CETSA) were performed to demonstrate the binding of FLT3 to GNF-7. The survival benefit of GNF-7 in vivo was assessed in mouse models of transformed Ba/F3 cells harboring FLT3-ITD and FLT3-ITD/F691L mutation. Primary patient samples and a patient-derived xenograft (PDX) model were also used to determine the efficacy of GNF-7.ResultsGNF-7 inhibited the cell proliferation of Ba/F3 cells expressing FLT3-ITD and exhibited potently anti-leukemia activity on primary FLT3-ITD AML samples. Moreover, GNF-7 could bind to FLT3 protein and inhibit the downstream signaling pathway activated by FLT3 including STAT5, PI3K/AKT and MAPK/ERK. In vitro and in vivo studies showed that GNF-7 exhibited potent inhibitory activity against FLT3-ITD/F691L that confers resistant to quizartinib (AC220) or gilteritinib. Importantly, GNF-7 showed potent cytotoxic effect on leukemic stem cells, significantly extend the survival of PDX model and exhibited similar therapy effect compared with gilteritinib.ConclusionsOur results show that GNF-7 is a potent FLT3-ITD inhibitor and may become a promising lead compound applied for treating some of the clinically drug resistant patients.

Intracellular IL-23 Receptor (IL-23R) Is a Regulator of Mitotic Spindle and Centrosome Formation and Is Essential for AML Viability

IL-23 receptor (IL-23R) is a heterodimeric cytokine cell surface receptor that is traditionally expressed on T cells. The cytokine, IL-23, is secreted by dendritic cells and macrophages in response to inflammatory stimuli. Here, we identify a novel function and localization for IL-23R in which intracellular IL-23R is necessary for AML viability and acts as a critical regulator of the mitotic spindle. We analyzed gene ontologies that were upregulated in AML samples compared to normal hematopoietic cells. Amongst upregulated gene ontologies, the mitotic spindle ontology was enriched in AML samples. Further analysis of all protein coding genes and their correlation to the mitotic spindle ontology surprisingly returned IL-23R as a top hit. Given that IL-23R is not known to be expressed in AML, we confirmed the presence of IL-23R protein by immunoblotting in 14 of 20 primary AML patient samples, while it was undetectable in bulk (n=5) and CD34+ sorted (n=3) normal hematopoietic cells. Consistent with previous investigations, we confirmed cell surface localization of IL-23R in double negative T cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were present on the cell surface of AML cells. Instead, IL-23R was detected intracellularly in the cytoplasm and nucleus of AML cell lines, as well as primary AML cells (including the stem cell fraction). We demonstrated intracellular localization using flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. To ensure we were detecting genuine IL-23R protein, we probed for IL-23R using 4 different antibodies targeting 4 different epitopes of the receptor whilst using 4 methods of detection (immunoblotting, flow cytometry, confocal microscopy, and immunoprecipitation). We also demonstrated intracellular localization of the IL-23R heterodimer subunit, IL12Rβ1, which is known to bind the IL-23R subunit to form the fully functional IL-23 receptor. To elucidate the function of intracellular IL-23R, we performed BioID mass spectrometry to identify proteins that interact with IL-23R. Compared to controls, we identified 61 proteins that preferentially interacted with IL-23R. 36 of those 61 proteins are known cytoplasmic or nuclear localized proteins. Pathway analysis of those interacting proteins identified the mitotic spindle as a top pathway corroborating with our bioinformatics analysis. Proximity Ligation Assay (PLA) and confocal microscopy verified that endogenous IL-23R protein interacted with mitotic spindle associated proteins, NUMA, TMEM201, TACC1, and BAG6 in OCI-AML2 cells and primary AML samples. Consistent with our PLA results, we demonstrated IL-23R co-localized with the mitotic spindle and centrosomes in AML cell lines and primary patient samples. Knockdown and knockout of IL-23R in AML cells led to the dysregulation of the mitotic spindle with multipolarity, lagging chromosomes, and spindle orientation errors. Knockdown of IL-23R reduced cell proliferation and viability in OCI-AML2, TEX, K562, NB4, and U937 cells compared to non-targeting controls. IL-23R knockdown also reduced the engraftment efficiency of TEX leukemia cells in murine bone marrow. Subsequent knockdowns of IL-23R in primary AML cells demonstrated decreased clonogenic growth and reduced engraftment into the marrow of immune deficient mice. In contrast, knockdown of IL-23R in normal human cord blood cells did not impair their engraftment into murine bone marrow. Finally, we analyzed hematopoietic cells and stem cells in IL-23R -/- mice and found that knockout of IL-23R did not decrease blood counts or the abundance and function of normal hematopoietic cells. In summary, we discovered a novel intracellular localization and function for IL-23R in AML. IL-23R regulates mitotic spindle and centrosome formation and is necessary for AML cell viability. We have thus identified a new biological function for IL-23R and a potential therapeutic target for AML.

The XPO1-FOXC1-HOX Functional Axis Opens New Therapeutic Avenues to Treat DEK-NUP214 AML Patients

Introduction: t(6;9)(p23;q34.1)/DEK-NUP214 patients represent a discrete group of younger AML patients recognised as a separate disease entity in the World Health Organization classification of myeloid neoplasms. They typically display a dismal prognosis, higher relapse rate and a striking co-occurrence with FLT3-ITD mutations in > 70% of cases. DEK is a nuclear factor that has been attributed multifunctional roles, including gene regulation, while its fusion partner, NUP214, plays a pivotal role in nuclear export by interacting with transport receptors such as XPO1. However, the precise mechanism by which DEK-NUP214 drives leukaemia remains unclear. Aims: Application of a multi-omics profiling to understand the oncogenic mechanisms underlying DEK-NUP214 fusion protein and identify potential therapeutical vulnerabilities. Methods: A comprehensive multi-omics screening (whole genome sequencing, targeted sequencing, transcriptomics, (phosphor)proteomics, mass cytometry (CyTOF) and in vitro drug screening) was conducted on 57 untreated primary AML samples from cytogenetically poor-risk AML, including four cases with t(6;9) translocation (complex karyotype (n=20), -7/del(7) (n=15), KMT2A rearrangements (not including t(9;11)) (n=12), t(6;9) (n=4) and other poor-risk karyotypes (inv(3), -5/del(5), t(3;12)/+8 or -17/del(17)) (n=6)). The t(6;9)/DEK-NUP214 cell line FKH-1, was utilised as a model for in vitro experiments and compared with other AML cell lines (OCI-AML3, KASUMI-1, THP-1, P31-FUJ, MV4-11 and K562). In vitro drug treatments were conducted in the cell lines and patient samples using Cell-Titer&Glo, and transcriptional changes were evaluated using qPCR and RNA-seq. To examine the function of selected target genes of DEK-NUP214, we used lentiviral shRNA system followed by cell cycle, apoptosis, differentiation, and cell proliferation assays. Results: An integrative RNA-seq analysis of our cohort and a separate series of 691 AML cases that included three additional t(6;9) cases (Leucegene, https://leucegene.ca/) revealed 128 genes significantly upregulated and 74 downregulated in t(6;9) patients compared to other AML samples. The overexpressed genes included some known leukaemia mediators (FOXC1, HOXA and HOXB genes), the genes previously reported in t(6;9) patients (EYA3, SESN1 and PRDM2) and novel genes with roles in haematopoiesis or cancer cell survival that showed the most significant differences (NFIX and GGT5). The significant overexpression of these genes was also confirmed in the t(6;9) FKH-1 cell line. In vitro drug screening of 527 licenced or investigational drugs allowed us to identify the compounds that were most effective and selective for t(6;9) patients in comparison with other AML cytogenetic groups. The two XPO1 inhibitors (Selinexor and Eltanexor) included in our drug panel were within the four top-ranked compounds. Of note, qPCR and RNA-seq analyses in the cell lines and primary patient samples showed that inhibition of XPO1 resulted in significant downregulation of the expression of FOXC1, NFIX, EYA3, HOXA and HOXB genes, highlighting a functional axis linking these target genes, XPO1 and the fusion. In line with this finding, silencing of FOXC1 in the FKH-1 cells led to an increase in apoptosis, lower cell proliferation and reduced clonogenic capacity, accompanied by a concomitant downregulation of HOXA and HOXB genes. Collectively, these findings support a key role of FOXC1 in DEK-NUP214-driven AML. Conclusion: This study offers valuable insights into the molecular and pathological mechanisms underlying t(6;9)-AML, revealing a functional interaction between DEK-NUP214, XPO1 and FOXC1, and providing evidence to consider XPO1 inhibition as a potential new avenue to treat these patients.

New Assay Technology and Utility of Peripheral Blood Vs Bone Marrow for Molecular MRD in AML and MDS

Background Rare tumor-specific mutations in patient samples serve as excellent markers to monitor the course of malignant disease and responses to therapy in clinical routine, and improved assay techniques are needed for broad adoption. We describe herein - superRCA assays - which provides for rapid and highly specific detection of DNA sequence variants present at very low frequencies in DNA samples using a standard flow cytometer for readout. We demonstrate and compare the assay against ddPCR for precise, ultra-sensitive detection of single-nucleotide mutant sequences from malignant cells to follow the course of AML and MDS patients following allogeneic stem cell transplantation, both in bone marrow and peripheral blood. Methods This study is based on patient material from the Nordic MDS study NMDSG14B (NCT02872662)and AML samples from the U-CAN biobank. The patients in this retrospective study have all previously undergone sequencing (TruSight panel, Illumina) upon diagnosis, after which patient specific driver mutations (1-4 per patient) has been chosen and analyzed longitudinally to monitor molecular MRD using ddPCR (Bio-Rad). These longitudinal samples were re-analyzed using superRCA mutation assays (Rarity Bioscience), a novel and ultra-sensitive technique for mutation detection using flow cytometer for readout. Both quarterly bone marrow aspirates and monthly peripheral blood samples post allogeneic stem cell transplantation were included to investigate to what extent frequent blood derived molecular MRD gives equivalent relapse information as bone marrow. For each mutation, sensitivity was assessed through serial dilution and wild type control, with the limit of detection calculated as Mean(wt)+3*SD(wt). Samples were analyzed in duplicates or triplicates and sample to sample variation was calculated as SD/Mean. Resul ts A total of 22 relapse patients and 21 different mutations were analyzed, covering single nucleotide variants, insertions, deletions, as well as mutations with very high GC in ASXL1 and SRSF2. The analysis demonstrating lower background and limit of detection for the superRCA assays compared to the equivalent ddPCR data. With spike-in dilution series, we demonstrated the superRCA assay could detect single point mutations as low as 1 in 100,000 with the flow cytometer readout. Mean background for superRCA was 0,0016% in wild type controls, and mean limit of detection was 0,0023%. From available serial dilutions, the mean sample to sample variation (CV) at 0,016% was 22,87% and 29,32% at 0,001% dilution. In longitudinal sample analysis of relapse patients, superRCA assay detected remaining mutations after initial treatment and clearly revealed the remaining malignant clone, subsequently leading to a relapse. The superRCA assay also demonstrated the feasibility of detecting corresponding leukemia mutations in the PBMCs when such mutations were present in bone marrow. Conclusions The data shows comparable mutant allele frequency results between superRCA and ddPCR upon diagnosis and relapse. The data further demonstrates superRCA mutation assays for the selected driver mutations can reach to very low limit of detection and increasing mutations could be detected in both blood and bone marrow predicting the relapse of MDS post transplantation in clinical samples. The low detection limit and high precision of superRCA are consequences of the highly selective genotyping of the repeated target sequences in combination with high input capacity and the large numbers of products that may be conveniently analyzed by flow cytometry. To further study the utility of using peripheral blood for molecular MRD in MDS and AML patients, more data is needed and studies are ongoing.

Identification of a Novel AML Patient Subset Sensitive to Venetoclax Using Matched Proteomics and Diverse Ex Vivo Models

AML is one of the most common and deadly hematopoietic malignancies. The molecular and biological heterogeneity of AML poses significant challenges to the identification of novel agents to treat it and the optimal triage of patients for the most effective therapeutic regimens. Ex vivo models for drug sensitivity using cell lines and/or primary AML samples have been a fruitful resource for the exploration and discovery of novel compounds in the treatment of AML, and investigators have coupled genomic and transcriptomic profiling with ex vivo models in the hope to further enhance the reliability and accuracy of results. However, primary AML blasts are molecularly complex and fastidious, relying on extracellular matrix (ECM) and signals for optimal survival, making their use for large, high-throughput drug screens impractical. Available AML cell lines are more robust and less dependent upon the ECM but often harbor strong driver alterations (e.g., KMT2A rearrangements, TP53 mutations) that occur in only a small subpopulation of AML cases. Thus, current AML cell lines may not accurately predict agent sensitivity for most patients. Here, we report the results of a novel approach coupling cutting-edge proteomics with ex vivo drug sensitivity models to develop and describe a more diverse and representative set of patient-derived cell models for drug discovery and therapeutic positioning. Initial studies focused on proteomic biomarker signatures and cell lines to predict drug sensitivity for Venetoclax, which has become a stable component for treatment of older and unfit patients with AML. To identify a proteomic biosignature associated with Venetoclax sensitivity, we examined the Beat AML dataset of 210 patient proteomics coupled to ex vivo sensitivity assays. Beat AML patients clustered by unbiased proteomics which resulted in a cluster with a patient population significantly enriched in sensitivity to Venetoclax. A partial least squares discriminant analysis (PLSDA) was used to discover proteins driving the differentiation of Venetoclax sensitive and insensitive clusters. The resulting significant proteins describe a mechanism of altered DNA double stranded break repair and Interleukin-8 (IL-8)-CXCR2 signaling separating patients. These mechanisms were partially comprised of high-quality targets with differential analysis results shown in Fig. 1A. The identities of the targets will be revealed at the time of presentation as they are currently moving forward in preclinical studies. To examine the potential predictive power of the Venetoclax sensitivity biosignature a set of novel ex vivo cell models were derived from a diverse independent population of patients obtained from FHCC/UW Myeloid Disease Repository. This population was not enriched for KMT2A or TP53 mutations. The proteomes of 90 diagnostic AML samples were examined and measured via a tandem mass tag protocol coupled with liquid chromatography with tandem mass spectrometry (TMT-LC-MS/MS). In addition, a proprietary set of novel ex vivo AML cell lines (n=17) representing the diverse genomic phenotype were generated from this same cohort of patients using proprietary media and pyrimidoindole derivative agonists of hematopoietic stem cell self-renewal. As with the primary samples, TMT-LC-MS/MS was utilized to quantify the proteome within the proprietary AML cell lines and 16 commercially available lines. Viability assays with Venetoclax were performed for all ex vivo cell models. Ex vivo cell models were then grouped based on the Venetoclax biosignature. The correlation between the proteomic biosignature for Venetoclax the measured sensitivity to Venetoclax was then examined for the ex vivo cell models (Fig. 1B). The results showed a strong correlation between the biosignature and actual ex vivo sensitivity to Venetoclax in proprietary cell models, but not in the 16 currently available cell models. Genetic drift of cell models from the primary AML patients was examined using differential proteomic analysis and no significant drift was found. The results indicated that the primary AML specimens harbored the Venetoclax sensitivity signature. Our findings reveal that the integration of unbiased patient proteomics with optimized ex vivo cell cultures can be used to identify sensitive patient subsets and exclude non-responders for novel therapeutics, as well as expanding patient access for currently approved therapies.

NK-2 Homeobox Transcription Factor NKX2-3 Confers Differentiation Block in NPM1-Mutated in Acute Myeloid Leukaemia

Dysregulation of HOX homeobox family transcription factor (TF) gene expression is important in the pathogenesis of AML in particular in NPM1-mutated and MLL rearranged molecular subtypes. However, little is known about the role of NK2-homeobox (NKX2) family TF genes. NKX2 genes have critical roles in normal development of the central nervous system, thyroid, lung and pancreas. We observed high expression of NKX2-3 in patient AML samples and hypothesised the transcription factor might have a functional role in the disease. By qPCR, we found that NKX2-3 is highly expressed in normal HSCs but is rapidly down regulated as cells differentiate. In primary AML samples, it is highly expressed in 21-37% cases, including within the immunophenotypic LSC compartment, in particular in cases with NPM1 and/or FLT3-ITD mutations. Forced expression of NKX2-3 in normal murine KIT + bone marrow (BM) HSPCs transiently enhanced their clonogenic activity and impaired differentiation: NKX2-3-expressing HSPCs expressed higher levels of E2F or MYC target genes, and leukemia stem cell (LSC) maintenance genes. Congenic transplant experiments revealed that forced expression of NKX2-3 in normal BM HSPCs was sufficient to enhance multilineage engraftment across four months of follow up without inducing leukemia. In keeping with this, transplantation of Nkx2-3 -/- knockout BM cells led to significantly inferior engraftment compared with BM cells from litter mate control mice. Thus, while increased expression of Nkx2-3 enhances engraftment of normal stem cells in transplantation assays, loss of Nkx2-3 impairs it. Analysis of NKX2-3 high primary human AML cases demonstrated that HOXA9 was the most highly upregulated transcription factor gene compared with NKX2-3 low cases. We expressed both factors singly and together in murine KIT+ BM HSPCs: Hoxa9/NKX2-3 cells exhibited significantly higher clonogenic activity and reduced morphologic and immunophenotypic differentiation compared with Hoxa9/MTV cells. Congenic BM transplant experiments demonstrated that Hoxa9/NKX2-3 recipients developed leukemias significantly earlier than Hoxa9/MTV recipients (median 82 vs 155 days). BM morphology and immunophenotyping confirmed that Hoxa9/NKX2-3AMLs exhibited a greater degree of differentiation block than Hoxa9/MTV AMLs. Leukemia cells from Hoxa9/NKX2-3 recipients coordinately expressed higher levels of E2F or MYC target genes, and LSC maintenance genes. NKX2-3 KD to ~30% of control values reduced proliferation and induced morphologic and immunophenotypic differentiation in primary patient NPM1-mutated AML cells. NKX2-3 KD led to down regulation of E2F and MYC target genes and normal human CD34 + stem cell genes; and up regulation of myeloid differentiation genes. Using a CRISPR ribonucleofection approach to relocate NPM1c to the nucleus we confirmed that NKX2-3 expression in NPM1-mutant primary AML cells is sustained by mutant NPM1c. ChIPseq in NPM1-mutated primary patient and KO52 AML cells identified NKX2-3 binding peaks. As expected, DNA sequences under peak apices were strongly enriched for NKX2-3 binding motifs; there was also significant enrichment for RUNX and ETS family motifs, but not HOX/MEIS/PBX motifs. To identify genes potentially regulated by NKX2-3, genomic coordinates of strong binding peaks were mapped to the single nearest gene. Remarkably, among genes up regulated following NKX2-3 knockdown in primary human AML cells there was a highly significant enrichment of genes with putative regulatory elements bound by NKX2-3. Likewise, among genes down regulated in NKX2-3-expressing KIT+ murine HSPCs, there was a similar significant enrichment of genes with putative regulatory elements bound by NKX2-3. These data suggest that NKX2-3 functions as a transcription repressor at bound regulatory elements in primary AML cells. In summary, NKX2-3 is highly expressed in NPM1 & FLT3 mutated molecular subtypes of AML and serves to enhance self-renewal and the level of differentiation block through binding to and repressing regulatory elements of myeloid differentiation genes.

Precise Single-Cell Transcriptomic Mapping of Leukemia Cell States Reveals Unconventional Lineage Priming in Acute Myeloid Leukemia

Initial disease classification within acute leukemia relies on identifying morphological and immunophenotypic features of hematopoietic differentiation retained by leukemic blasts. While existing approaches can classify leukemic cells into broad lineages, they lack precision in discerning between specific cell states, particularly at the level of immature blasts. Single-cell (sc) RNA-sequencing (RNA-seq) provides thousands of new markers to enable precise determination of leukemia cell state and provides an opportunity to refine our classification of acute leukemia. To extend our ability to identify leukemia cell states by scRNA-seq, we developed a comprehensive reference map of human bone marrow hematopoiesis. Specifically, we integrated three unsorted bone marrow datasets and three CD34+ hematopoietic stem and progenitor (HSPC) purified datasets to ensure balanced representation of early HSPCs and terminally differentiated immune cells. This culminated in a curated atlas of 263,159 high-quality sc transcriptomes, representing 55 cellular states spanning 45 healthy donors (Fig 1A). We validated our cell state annotations to ensure concordance between transcriptional states and functional populations through reference mapping of bulk and sc transcriptomes from purified HSPC subsets. For example, mapping of scRNA-seq profiles from Lin-CD34+38-45RA-90+49f+ long-term (LT) HSCs revealed 91% concordance between transcriptional HSC and functional LT-HSC. We next performed scRNA-seq profiling on 12 AML patient samples harbouring either myelodysplasia-related genetic features or classical alterations involving NPM1 or KMT2A. By mapping leukemia cells onto our reference atlas, we identified pronounced involvement of early erythroid-like blasts in a subset of AML samples. To further investigate unconventional lineage priming in AML, we processed and mapped scRNA-seq profiles from eleven studies comprising 166 additional AML samples and 9 mixed phenotype acute leukemia (MPAL) samples (Fig 1B). After quality control and exclusion of mature lymphoid cells, we performed composition analysis utilizing 600,570 leukemia cells mapped to 38 cell states to identify patterns of variation among leukemia cell states. Immature leukemia cells map to a range of cellular states spanning HSC/MPP-like, LMPP-like, MLP-like, GMP-like, MEP-like, and EarlyEry-like, among others. Notably, we identified a subset of AMLs with high MLP-like enrichment that co-cluster with MPALs as well as a subset with high EarlyEry-like involvement that co-cluster with acute erythroid leukemias (AEL), thus highlighting the biological continuum between these diseases. We next identified marker genes from each leukemia cell state and trained sparse regression models to estimate their relative abundance within bulk RNA-seq cohorts. Through bulk analysis, we confirmed that MPAL is highly enriched for MLP-like cells compared to AML (p=2.5e-12) and that M6 AELs are highly enriched for EarlyEry-like cells compared to M0-M5 AMLs (p=0.00067). Among 864 AML patients, we found that high MLP-like abundance was associated with NUP98-NSD1 fusions (p=0.0011) and RUNX1 mutations (p=8.0e-10) while high EarlyEry-like abundance was associated with Complex Cytogenetics (p=8e-15) and TP53 mutations (p=2.3e-25). Cellular states of immature leukemic cells captured inter-patient heterogeneity within 136 AEL samples, wherein high EarlyEry-like abundance was associated with TP53 mutations (p=3.6e-9) and Poor cytogenetic risk (p=1.1e-9) while high GMP-like abundance was associated with KMT2A alterations (p=0.00046) and Good/Intermediate risk (p=0.00032). Finally, through integration of sc transcriptomics and targeted DNA profiling, we found examples of genetic subclones impacting the cellular hierarchies of individual patients through induction of early differentiation blocks, skewing of lineage output from myeloid to erythroid, or potentiation of transcriptional self-renewal programs within mature myeloid cells. Together, our high-resolution mapping approach highlights the cellular state continuum between acute leukemias and enumerates the impact of genetic drivers on leukemia cell hierarchies. Precise definitions of cellular states, coupled with the presence or absence of genetic alterations, may help to refine future disease classification, prognostication, and therapy development.

Long-Acting E. coli-Derived Asparaginase Potentiates the Anti-Leukemic Effect of BCL2 Inhibition, but Not MCL1 Inhibition, in Preclinical Models of Acute Myeloid Leukemia

Background: Asparaginases are enzymes that deplete circulating asparagine and glutamine from plasma and can therefore be used therapeutically for cancers that are sensitive to amino acid depletion. Commercially available asparaginases are isolated fromeither E. coli or Erwinia chrysanthemi (also called crisantaspase), the latter having a higher glutaminase activity. Our previous studies have shown that pegcrisantaspase (PegC), a long-acting pegylated crisantaspase, synergizes with the BCL2 inhibitor, Venetoclax (Ven), to kill several AML cell lines and patient-derived primary AML cells with complex karyotype in vitro and in vivo in a xenograft mouse model. Whether long-acting E. coli-derived asparaginases will have similar anti-AML activity as a single agent or in combination with BCL-2 inhibition, and the potential of asparaginases to synergize with inhibitors of MCL1, a key resistance factor of BCL2 inhibition, has yet to be determined. Methods: Using a panel of 4 human AML cell lines (MOLM14, MV411, MonoMac6, HL60) as well as primary AML patient samples, weestablished the anti-leukemic activity of the BCL2 inhibitor S55746 and the MCL1 inhibitor S63845 alone and in combination with the long-acting E. coli asparaginase, calaspargase pegol-mknl (CalPegA). To determine the ability of S55746 or S63845 with or without CalPegA to inhibit mRNA translation, an established mechanism of Ven-PegC anti-leukemic activity, we measured the dissociation of the translation initiation factor eIF4E from 4EBP1, a negative regulator of cap-dependent mRNA translation, which then allows the formation of translation initiation complexes. The in vivo anti-leukemic efficacy of S55746 and S63845 alone and in combination with CalPegA was tested using an orthotopic AML xenograft mouse model with luciferase-expressing MV411 cells. Results: We found that CalPegA treatment resulted in a dose-dependent inhibition of AML cell proliferation, with IC 50 values ranging from 0.78 to 2.8 IU/mL. Single agent S55746 and S63845 treatment inhibited AML cell proliferation with IC 50 values ranging from 0.01 to 6.7 µM and 0.0001 to 0.08 µM, respectively. In 3 of the human AML cell lines (MOLM-14, MV4-11, and MonoMac6), the addition of a small dose (IC 20) of CalPegA potentiated the anti-leukemic effect of S55746, reducing the IC 50 by approximately 1- 4-fold, and by 10-fold in HL60 cells (Figure 1). This potentiation was also observed in 3 out of 4 primary AML patient samples tested, with the IC 50 of S55746 reduced by 4.5, 18, and 156-fold by the addition of CalPegA. The activity of S63845 was not substantially impacted by the addition of CalPegA in either AML cell lines (Figure 1) or primary samples. Immunoprecipitation of 4EBP1 revealed that treatment with a combination of either S63845 or S55746 with CalPegA increased the binding of eIF4E to 4EBP1 compared to single agent treatments, suggesting an inhibition of translational complex formation. In a MV411 tumor-bearing model, treatment withS55746-CalPegA combination reduced tumor burden over time significantly more than either single agent. S63845 treatment was highly effective at inhibiting leukemia growth over time and was not improved by the addition of CalPegA, as tumor burden in the S63845 and S63845 + CalPegA-treated groups was not significantly different over the course of the study (Figure 2). Conclusions: We report that the E. coli asparaginase, CalPegA, enhances the anti-leukemic effect of the BCL2 inhibitor, S55746, but does not impact the activity of the MCL1 inhibitor, S63845, in AML cell lines, patient derived primary AML samples, and in an AML xenograft mouse model. Ongoing studies are further investigating the anti-leukemic mechanism of S55476/S63845 + CalPegA and examining the efficacy and pharmacodynamic/pharmacokinetic effects of these agents in a patient derived xenograft model of AML.