KMT2A-rearranged Acute Myeloid Leukemia Research Articles

KRAS G12 mutations as adverse prognostic factors in KMT2A-rearranged acute myeloid leukemia.

KRAS G12 mutations as adverse prognostic factors in KMT2A-rearranged acute myeloid leukemia.

Abstract 5592: HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia

Abstract Although High Mobility Group A1 (HMGA1) chromatin regulators are overexpressed in refractory acute myeloid leukemia (AML) and diverse solid tumors, targetable mechanisms underlying HMGA1 remain elusive. HMGA1 is highly expressed in stem cells and poorly differentiated cancers where it modulates chromatin structure to activate developmental gene networks. In AML arising from JAK2V617F myeloproliferative neoplasms (MPN), HMGA1 is required for leukemogenesis by inducing transcriptional networks that drive aberrant proliferation and cell fate. Like MPN AML, KMT2A-rearranged (KMT2A-r) AML is refractory to therapy and highly lethal. KMT2A-r AML is caused by rearrangements of the KMT2A gene, which encode abnormal fusion proteins that induce pro-leukemogenic genes, including HOX genes. While prior work identified KMT2A fusion partners and protein complexes, this has not led to better therapies for most patients. We therefore sought to examine HMGA1 function in KMT2A-r AML. We hypothesized that: 1) HMGA1 drives plasticity in KMT2A-r AML by altering chromatin state and transcriptional networks, and, 2) Targeting HMGA1 will enhance sensitivity to therapy. In two KMT2A-r AML patient cohorts, HMGA1 is highly overexpressed compared to CD34+ stem and progenitors from healthy controls, suggesting it drives leukemogenesis. To define HMGA1 function, we silenced HMGA1 using CRISPR or short hairpin RNA in KMT2A-r cell lines (MOLM-14, THP-1, MV4-11). Strikingly, silencing HMGA1 disrupts proliferation and clonogenicity in vitro, while prolonging survival in immunosuppressed mice transplanted with KMT2A-r AML cells. Intriguingly, leukemic cells that engraft in the bone marrow from the pool of cells with HMGA1 silencing express higher levels of HMGA1, suggesting that escape from gene silencing and a specific level of HMGA1 is required for leukemic engraftment and expansion. HMGA1 also increases in KMT2A-r AML cells that become resistant to cytarabine (AraC), suggesting that HMGA1 endows AML cells with the capacity to survive and expand after AraC. Because repression of tumor suppressors was recently defined as a mechanism of therapy resistance in AML, we also examined tumor suppressor pathways in both JAK2V617F and KMT2A-r AML. Intriguingly, HMGA1 occupies the promoter region of the CDKN1A locus by chromatin immunoprecipitation and represses its expression in KMT2A-r and JAK2V617F AML cells. To define additional mechanisms underlying HMGA1 in KMT2A-r AML, multi-omics studies are underway. In preliminary work thus far, we also found that HMGA1 induces HOX genes. Together, these findings implicate HMGA1 as a promising therapeutic target and novel epigenetic regulator in KMT2A-r AML by altering chromatin state and downstream gene expression. Citation Format: Bailey E. West, Jung-Hyun Kim, Audrey-Ann Supreme, Iliana Herrera, Zanshé Thompson, Li Z. Luo, Faiza Shaik, Joseph Kim, Hyunsung Woo, Soheil Meshinchi, Rhonda E. Ries, Linda M. Resar. HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia [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 5592.

Abstract 3218: Combination drug screen identifies novel epigenetic therapies to enhance menin inhibitor efficacy in KMT2A rearranged and NPM1 mutant AML

Abstract Background: Acute myeloid leukemia (AML) with KMT2A-rearrangements have a poor prognosis and so there is an urgent need for novel therapies. Menin inhibitors have shown promising anti-leukemic effects in KMT2A-rearranged and NPM1-mutated AML. However, the efficacy of menin inhibitors as single agents may be limited by the development of resistance mutations. Combining menin inhibitors with other epigenetic drugs may present a strategy to enhance their therapeutic potential. In this study, we performed a combination drug screen using an epigenetic drug library in KMT2A-rearranged (MV4;11) and NPM1-mutant (OCI-AML3) AML cell lines to identify synergistic therapies that can be used in conjunction with menin inhibitors. Methods: During assay optimization, MV4;11 and OCI-AML3 cells were treated with the menin inhibitor DSP-5336, and their viability was assessed. Quality control metrics were applied with Z’ values consistently greater than 0.5 for all assay plates and then the cells were subjected to a small molecule compound library screen containing a set of 932 epigenetic drugs. The screening process included a systematic evaluation of each drug's effect on cell viability in combination with the DSP-5336. Results: The combination drug screen demonstrated robust performance in both MV4;11 and OCI-AML3 cell lines, with reliable quality control metrics. Among the epigenetic drugs tested, five compounds were identified as potential hits that synergistically enhanced the efficacy of DSP-5336 across both cell lines.The five identified compounds exhibited enhanced anti-leukemic effects when combined with the menin inhibitor in either one of the cell lines. The targets of these drugs varied, including histone deacetylases, histone acetyltransferases and BRD4 inhibition, suggesting many alternate mechanisms of action involving epigenetic modulation. Remarkably, one of the 5 hits C301-3161, a KAT2A inhibitor, showed limited activity in either cell line alone but increased cell killing with DSP-5336 in both cell lines, highlighting its potential as a specific therapeutic for combining with DSP-5336 in AML patients. Conclusions: Our findings suggest combining menin inhibitors with other epigenetic drugs may improve AML treatment outcomes. The combination drug screen successfully identified four novel compounds that synergistically enhance menin inhibitor efficacy in both KMT2A-rearranged and NPM1-mutant AML cell lines. These compounds hold significant promise as potential therapeutic candidates for further investigation. Future studies should focus on elucidating the underlying mechanisms of action for each hit and evaluating their effects in preclinical animal models. Ultimately, this research may pave the way for the development of innovative combination therapeutic strategies for AML patients with KMT2A-rearragned and NPM1-mutant AML. Citation Format: Tulasigeri M. Totiger, Sana Chaudhry, Skye Montoya, Maurizio Affer, Anya K. Sondhi, Alexandra Chirino, Claude-Henry Volmar, Shaun Brothers, Justin Watts, Justin Taylor. Combination drug screen identifies novel epigenetic therapies to enhance menin inhibitor efficacy in KMT2A rearranged and NPM1 mutant 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 3218.

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

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

Genomic Analyses Unveil the Pathogenesis and Inform on Therapeutic Targeting in KMT2A-PTD AML

A partial tandem duplication (PTD) in the KMT2A gene is detected in approximately 5-10% of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Previous studies revealed that HOX-genes are highly expressed in KMT2A-PTD AML similar to KMT2A-rearranged or NPM1-mutated AML. While the overexpression of HOX-genes is a convergent oncogenic pathway shared by these molecular subtypes of AML, the mechanism by which KMT2A-PTD induces high expression of the HOX-genes remains unclear and may be distinct from that mediated by balanced KMT2A-translocations, and NPM1 mutations. We stipulated that analysis of common molecular features of KMT2A-PTD will help identifying mechanistic intersections pointing towards possible targeted therapies. In a total of 5420 AML cases 1 including 793 cases from publicly available sources (Beat AML 2 and TCGA 3), we identified 254 cases of KMT2A-PTD. Somatic mutational screening and karyotype analysis showed co-occurrence with DNMT3A (31%), FLT3-ITD (32%), IDH1/2 (26%), RUNX1 (28%), TET2 (18%), SRSF2, STAG2, U2AF1 mutations and deletion 5q. Strong leukemic initiators such as KMT2A-rearrangement, CBFB-MYH11, PML-RARA, RUNX1-RUNX1T1, trisomy 8 and NPM1 mutations were mutually exclusive with KMT2A-PTD and did not rely on acquisition of additional mutations linked to secondary AML ontogenesis ( e.g., ASXL1, RUNX1, SRSF2, and STAG2) different from KMT2A-PTD. Next, we analyzed bulk mRNA-expression between mutually exclusive KMT2A-PTD, KMT2A-rearranged or NPM1-mutant high HOX-genes expressors. While several differentially expressed genes (DEG) between KMT2A-PTD and KMT2A-rearranged cases were detected, there were few DEG between KMT2A-PTD and NPM1-mutated cases. Compared to KMT2A-rearrenged cases, KMT2A-PTD had higher expression of CD34 and KIT in addition to some HOX-genes such as HOXB2 or NKX2-3. In contrast, expression of CD1D, CD14, and immune checkpoint genes including LILRB4, CD276, CD86, and SIGLEC7 was much higher in KMT2A-rearranged than KMT2A-PTD cases. These results prompted us to analyze the hematopoietic differentiation status. For this purpose, we defined three gene signatures along the HOX-targeted genes axis, termed as HOX-primitive, HOX-transient, and HOX-committed profiles. Differentiation analysis revealed almost all of KMT2A-PTD cases could be subclassified into HOX-transient stage while most of KMT2A-rearranged cases were HOX-committed. However, NPM1-mutated cases were interspersed in all the differentiation stages along the HOX-axis. We therefore assumed that there were a few DEG shared between KMT2A-PTD and NPM1-mutated cases as some NPM1-mutated AML clustered in the HOX-transient expression profile group. Intriguingly, DEG analysis was concordant with the hierarchical clustering based on the HOX-gens axis. Indeed, CD34 and KIT were highly expressed in the HOX-transient cases, and CD1D and CD14 in the HOX-committed cases, consistent with the enrichment of KMT2A-PTD and KMT2A-rearranged AML, respectively. We can conclude that the observed signatures were unique to the HOX-stages rather than the molecular abnormalities such as KMT2A-PTD and KMT2A-rearrangements. For example, CD34 upregulation was a feature shared transversally by all HOX-transient cases, including both KMT2A-rearranged and NPM1-mutated cases. As shown in Figure, KMT2A-rearranged, KMT2A-PTD, and NPM1-mutated cases were more clearly classified in HOX-profiles than molecular abnormalities. Additionally, among the cases which molecular subtypes were not clear, those with high HOX-genes expression revealed higher MEN1 expression. In conclusion, despite molecularly different, KMT2A-PTD, KMT2A-rearranged, and NPM1-mutated AML have similar expression patterns defined by each HOX differentiation stage. Like core binding factor AML such as RUNX1-RUNX1T1 and CBFB-MYH11, AML with higher expression of HOX-genes should be classified in the same group, HOX-AML. Given the results obtained with menin inhibitors in KMT2A-rearranged and NPM1-mutated AML, our findings open an opportunity for exploiting a therapeutic vulnerability in all HOX-AML including KMT2A-PTD AML or AML with high MEN1 expression. Since HOX-AML highly express genes according to the HOX differentiation profile, stage-specific surface proteins coded by these genes would be promising targets.

Integrated Methylation and Transcriptional Landscape and Evolution of Pediatric AML

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy associated with poor outcomes despite intensive therapies. The genomic landscape of pediatric AML has been well characterized and has led to translational benefits, such as improved relapse predication and effective targeted therapies for a subset of individuals (e.g., FLT3-ITD AML). Despite these advances, the majority of patients continue to receive therapeutic interventions that have not changed in three decades. This underscores the need to advance our understanding of AML biology beyond genomic alterations (e.g., somatic mutations). Here, we present the largest (N = 858 patients) integrative analysis of pediatric AML paired transcriptomes and methylomes to date, performed at diagnosis for patients enrolled on Children's Oncology Group clinical trials AAML0531 (NCT00372593) or AAML1031 (NCT01371981). We also analyzed N = 400 and 253 corresponding transcriptomes and methylomes at relapse, respectively. Additionally, we performed comprehensive mutation and fusion calling from NGS data and integrated clinical covariates and outcomes. This robust dataset allowed us to uncover how pediatric AML epigenetics are perturbed from normal hematopoiesis, characterize the evolution of transcriptional and methylation changes from leukemia diagnosis to relapse, and identify more accurate relapse prediction models for pediatric AML. We initially performed unbiased hierarchical clustering on CpG sites that are differentially methylated between AML and normal bone marrow to identify distinct AML-restricted methylation (ARM) signatures. This analysis demonstrated that that these ARM clusters were enriched in specific cytomolecular subtypes of pediatric AML. Specifically, we identified methylation signatures that recapitulated their adult counterparts (e.g., IDH1/2, DNMT3A, WT1) and pediatric specific methylation signatures that clustered with RUNX1::RUNX1T1, CBFB::MYH11, KMT2A-rearrangement, NUP98::NSD1, NUP98::KDM5A, and CEBPA mutant AML. Next, we investigated the transcriptional and methylation signatures for the relapsed cases and compared them to diagnosis. We demonstrated that relapsed transcriptional and methylation signatures clustered within their cytomolecular subtype counterparts at diagnosis (i.e., AMLs at relapse more closely resemble AMLs at diagnosis with the same underlying gene fusion than other relapsed AMLs) ( Fig. 1A). However, analyzing our data in the context of these cytomolecular subtypes, we found that leukemias uniformly acquired a more stem-like state at relapse. We focused our biomarker discovery efforts on KMT2A-rearranged AMLs since (as a cohort) these patients have an intermediate risk of relapse and existing molecular biomarkers (e.g., KMT2A fusion partner and measurable residual disease detection) result in only modest improvements in relapse prediction. First, we found that CpG islands associated with CD34 and HOXA gene expression were frequently (but variably) hypermethylated, which was also associated with downregulation of CD34 and HOXA gene expression and favorable survival outcomes. Next, we performed non-negative matrix factorization on diagnosis and relapsed KMT2A-rearranged AMLs and identified a novel gene set associated with relapse and poor outcomes. These three distinct methylation-transcriptional findings (i.e., CD34, HOXA, and relapsed signatures) were subsequently transformed into a novel methylation-transcription biomarker score (MTBS) using machine learning algorithms (i.e., LASSO Cox regression). We used KMT2A-rearranged AML patients from AAML1031 as our training cohort (N = 129) to generate a MTBS that was able to distinguish KMT2A-rearranged cases with disparate outcomes. This algorithm was validated in an independent cohort from AAML0531 (N = 28) ( Fig. 1B). Additional methylation-transcriptional biomarker discovery efforts for pediatric AML are underway. Since pediatric AML is associated with a low mutational burden, understanding leukemia-specific transcriptional and epigenetic alterations is of utmost importance. Our study demonstrates the translational promise offered by deep functional genomic characterization of pediatric AML and we believe that further interrogation may provide additional novel insights into leukemia initiation, relapse prediction, and novel treatment approaches.

Decoding the Epigenetic Drivers of Menin-MLL Inhibitor Resistance in KMT2A-Rearranged Acute Myeloid Leukemia

Menin inhibitors that disrupt Menin-MLL interaction are candidate therapeutic agents for several acute myeloid leukemia (AML) subtypes such as KMT2A rearrangement and NPM1 mutation. Several Menin inhibitors are currently being assessed in clinical trials and have demonstrated promising initial results. However, the development of both innate and acquired resistance to Menin inhibitors could limit their therapeutic potential. While studies have found that around 40% of these resistance cases can be traced back to mutations in the Menin gene, the Menin gene remains unaltered in other majorities, suggesting the presence of other mechanisms driving resistance. Here, we developed a unique chromatin-focused CRISPR knockout library, named Advanced Catalogue of Epigenetic Regulators (ACER). The ACER library contains approximately 800 traditional and newly-predicted epigenetic regulators using epigenetic network analyses that integrated publicly available gene codependency (DepMap) and protein-protein interaction data (BioPlex) (Figure A). To understand the chromatin mechanisms responsible for Menin-MLL inhibition resistance, we employed the ACER library for CRISPR screen in the context of Menin inhibition. Notably, our screen identified non-canonical polycomb repressive complex PRC1.1 (PCGF1, BCOR, and RYBP) and PRC2.2 (EZH2, SUZ12, and EED) among the top hits that conferred resistance to MLL-Menin inhibition upon gene depletion (Figure B). Consistent with our screen results, the depletion of PRC1.1 components led to a markedly increase in IC50 values when treated with the Menin inhibitor VTP50469 in both human and murine KMT2A-rearranged cell models. While treatment with Menin inhibitors typically induces cellular differentiation in leukemia cells, depletion of PRC1.1 genes resulted in an almost complete blockage in this differentiation process. Menin inhibitors evict Menin from chromatin and repress the expression levels of Menin-MLL target genes like MEIS1, PBX3, and MEF2C. Strikingly, our ChIP-seq and RNA-seq data indicatethat the loss of PRC1.1 components doesn't impact Menin's displacement from chromatin or the repression of KMT2A target genes by the Menin inhibitor. This is consistent with recent data from phase I clinical trial of revumenib (SNDX-5613) and preclinical patient derived xenograft models, wherein the Menin inhibitor persistently inhibitedkey KMT2A targets, even in resistant samples. These findings highlightthe possibility that resistance to Menin inhibition arising from mechanisms independent of canonical KMT2A target genes. Importantly, our unbiased transcriptomic analysis revealed that MYC signatures as the most affected pathway in PRC1.1-depleted cells following Menin inhibition. Furthermore, we show that both the transcriptionally active Menin-MLL and the repressive PRC1.1 complexes coexist at the MYC promoter, and the interplay between these two complexes determines MYC transcription output in AML. Moreover, enforced expression of MYC leads to resistance against Menin inhibition, while genetic and pharmacologic targeting MYC in PRC1.1 deficient cells rescued the resistance to Menin inhibition. Finally, in our RNA-seq datasets, we observed a diminished monocyte differentiation signature after PRC1.1 depletion. Considering AML cells with monocytic signatures, typically seen in KMT2A-rearranged AML, often exhibit resistance to BCL-2 inhibitor venetoclax, we hypothesized that AML cells lacking PRC1.1 could be more susceptible to BCL2 inhibition. This was confirmed in our ACER library screen with venetoclax treatment, where the PRC1.1 components PCGF1 and BCOR stood out as top epigenetic factors rendering drug sensitivity. Our subsequent studies using murine AML models and human primary samples further validated that PRC1.1-deficient AML cells are markedly sensitive to venetoclax. Furthermore, we show that venetoclax can be employed to overcome the resistance to Menin-MLL inhibition. In summary, our study identifiesthe non-canonical PRC1.1 as a key epigenetic driver of Menin-MLL resistance through a KMT2A target gene-independent mechanism which involves aberrant activation of MYC. We have further provided evidence that AML cells with loss of PRC1.1 are hypersensitive to BCL-2 inhibitor Venetoclax, opening a new therapeutic avenue for tackling Menin-resistant AMLs driven by polycomb inactivation.

CD276 (B7-H3) Is an Immunotherapeutic Target in Acute Myeloid Leukemia with Preclinical Efficacy of Vobramitamab Duocarmazine, an Investigational CD276 Antibody-Drug Conjugate

CD276 (B7-H3) is an immune checkpoint inhibitor expressed in a majority of solid tumors and hematologic malignancies, however it has not been well-described in acute myeloid leukemia (AML). A variety of immunotherapies targeting CD276 including CAR T cells and antibody-drug conjugates (ADCs) are being explored in solid tumors, however their role in leukemia has not been well elucidated. We interrogated the transcriptome and associated clinical and laboratory data from a large cohort of pediatric and adult AML patients to define the prevalence, frequency of co-occurring genetic alterations and outcomes associated with CD276 expression. Lastly, we evaluated preclinical in vitro efficacy in AML of vobramitamab duocarmazine (vobra duo), an investigational ADC with a humanized B7-H3 mAb conjugated via a cleavable linker to the prodrug seco-DUocarmycin hydroxyBenzamide Azaindole. Ribo-depleted RNA sequencing data from 2,241 patients with AML (Pediatric ages 0-29 years N=1,412, from recent Phase 3 COG clinical trials; Adult ages 18-88 years BEAT AML N=451, SWOG N=199, TCGA-LAML N=179) with available cytogenetic, molecular, and clinical data were utilized. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytomolecular aberrations. Response was measured by 5-year overall survival (OS), event-free survival (EFS) and relapse risk (RR). Of the 1,412 pediatric patients evaluated, CD276 mRNA expression was positive (defined as mRNA transcripts per million (TPM) &amp;gt;2) in 289 patients (20%). Higher prevalence (58%) was seen in adult AML patients (BEAT AML N=209/451, SWOG N=191/199, TCGA-LAML N=85/179), Fig 1A. Flow cytometry validated cell surface expression in pediatric AML, with 22% of patients expressing CD276. Cell surface expression was also observed in AML cell lines. CD276 mRNA expression is absent in normal hematopoietic cells (TPM &amp;lt;1) with absent cell surface expression in normal bone marrow as assessed by flow cytometry, allowing for targeted CD276 therapies to be utilized with no/minimal on-target off-tumor hematopoietic toxicity. Evaluation of co-occurring cytomolecular aberrations demonstrated a significant enrichment of CD276+ pediatric patients with high-risk genetic alterations, most notably KMT2A fusions (N=150/343, 43.7%), KAT6A-CREBBP (N=11/12, 91.6%) and CBFA2T3-GLIS2 (N=22/39, 56.4%). We evaluated the significance of CD276 mRNA expression on clinical outcomes in childhood AML. 5 year event free survival (EFS) for patients with CD276 expression was 35% compared to 44% for patients without CD276 expression (p=0.0022). Overall survival (OS) was 52% for patients with CD276 expression compared to 61% for patients without CD276 expression (p=0.0015). Relapse was seen in 40.5% of patients with CD276 expression compared to 35.8% of patients without CD276 expression and relapse occurred on average earlier in CD276 positive patients (0.89 years vs. 1.06 years, p=0.02). Given the poor clinical outcomes for patients with high CD276 expression, novel targeted therapies are needed for this high risk cohort. With multiple CD276 immunotherapies under clinical evaluation in solid tumors, we tested vobra duo in OCI-AML2, a CD276 positive KMT2A-rearranged AML cell line, which demonstrated robust cytolytic activity with an IC50 less than 1nM (IC50=0.9306 nM) supporting the potential efficacy of CD276 targeted therapies in AML (Fig 1B). In a large cohort of AML patients, we identified CD276 to be overexpressed in 20% of pediatric AML patients and 58% of adult AML patients with a significant enrichment in high risk fusion subtypes including KMT2A-rearranged AML, KAT6A-CREBBP and CBFA2T3-GLIS2 fusions. Patients with CD276 expression have poor outcomes with significantly worse EFS and OS compared to patients who do not have CD276 expression. With multiple CD276 immunotherapies under clinical evaluation for solid tumors, we hypothesized that targeting CD276 in AML would result in specific cytolytic activity. Vobra duo showed robust in vitro cytolytic activity against CD276 positive AML cells highlighting the need for ongoing preclinical evaluations of CD276 targeted therapies in AML. Given the established safety profile for vobra duo this provides a clear path for rapid translation to clinical use for high risk AML patients.

Potent and Target Specific Cytotoxicity of PRAME TCR Mimic CAR T Cells in KMT2A-Rearranged AML Patient-Derived Xenograft Model

Chimeric antigen receptor (CAR) T cell therapy was a resounding success in CD19+ ALL despite the fact that it is essential for B-cell maturation because it is not expressed during normal myeloid hematopoiesis. Normal and leukemic B-cells are targeted by CD19+ CAR T-cells without causing myeloid hematopoietic toxicity. Advancing such therapy to acute myeloid leukemia (AML) has been challenging in part due to a paucity of “dispensable” targets or targets whose expression is limited to the leukemic cells. Using a large AML and normal hematopoiesis transcriptome database, we pursued discovery of “AML-Restricted Targets”; genes that are silent in normal hematopoiesis but expressed in AML. This broad discovery effort yielded a library of AML-restricted targets that included PRAME (Preferentially Expressed Antigen in Melanoma), an intracellular protein expressed via MHC on the cell surface, to be a highly expressed in AML including the aggressive KMT2A-rearranged (KMT2A-r) AML. We used a TCR mimic (mTCR) antibody, which recognizes the PRAME peptide/HLA-A2 complex on the tumor cell surface, to develop a PRAME directed TCR mimic CAR T (PRAME mTCRCAR T) for pre-clinical studies. Studies in cell lines and CDX models have shown significant efficacy of this CAR T (Kirkey, Bld Adv. 2022). To conduct final IND-enabling studies, we generated a KMT2A-r HLA-A2+/PRAME+ patient-derived xenograft (PDX) model to treat with PRAME mTCRCAR T-cells. Here we demonstrate the in vivo activity of PRAME mTCRCAR T cells against this unique PDX AML model. VL and VH sequences from the PRAME specific TCR mimic antibody (Pr20) were used to construct the single-chain fragment variable domain into the 41-BB/CD3ζ CAR vector. The PDX model was derived from a PRAME +/HLA-A2 + pediatric patient with KMTA2-r AML. PDX cells were transduced with lentiviral ffluciferase for noninvasive bioluminescent IVIS imaging to monitor leukemic progression. Mice were transplanted with 1x10 6 PDX leukemia cells then 1 week later, PDX leukemia-bearing mice were treated with unmodified T cells or PRAME mTCRCAR T cells at 5x10 6 cells (1:1 CD4:CD8) per mouse. Leukemia burden was measured by IVIS imaging and regular peripheral blood analysis. Mice who received unmodified T cells had rapid disease progression by day 50 and all died by day 105. In contrast, PRAME mTCRCAR T-treated mice rapidly cleared disease and all remained alive and leukemia-free &amp;gt;180 days post-treatment (p=0.001). Average leukemia burden in the control cohort was 3.5% at week 6 and 41.75% at week 11, while no leukemia was detected following CAR T-cell treatment. In addition, the control arm had a marked expansion of leukemia with a 7.75- and 429.6-fold increase in radiance by IVIS at 6 and 11 weeks post-treatment, respectively. No significant increase in radiance was seen in the PRAME mTCRCAR T cell treated group. We further evaluated hematopoietic toxicity of PRAME mTCRCAR T cells in humanized mice. Sub-lethally irradiated NSG-SGM3 mice were reconstituted with HLA-A2+ CD34 selected human cord blood stem cells with simultaneous and allowed to engraft. Human hematopoietic cell engraftment was evaluated following treatment with unmodified T cells vs. PRAME mTCRCAR T cells. Evaluation of human CD45+ cells in the peripheral blood via flow cytometry showed no difference in the levels of hematopoietic engraftment in mice treated with PRAME mTCRCAR T cell (8%) vs. unmodified T cells (6%) vs. an untreated control cohort (5%), demonstrating lack of toxicity of PRAME mTCRCAR T cells against HLA-A2 hematopoiesis. We demonstrate that PRAME mTCRCAR T cells can irradicate AML in a target specific manner in aggressive KMT2A-r AML without toxicity. We show potent efficacy with eradication of leukemia in PDX-bearing mice following treatment with PRAME mTCRCAR T cells resulting in prolonged survival, without hematopoietic toxicity. These results provide strong rationale for advancing this therapeutic approach to clinical development.

Chip-AML22 Master Protocol: An Open-Label Clinical Trial in Newly Diagnosed Pediatric De Novo Acute Myeloid Leukemia (AML) Patients Including a Linked Phase II Trial with Quizartinib in FLT3-ITD/ NPM1wt Patients - a Study By the NOPHO-DB-SHIP Consortium

Background and Significance Pediatric AML is a heterogeneous disease with current rates for event-free survival (EFS) of 50-60% and for overall survival (OS) of 70-80%, for newly diagnosed patients. In patients with refractory and relapsed disease, and in subsets as defined by AML biology and treatment response, prognosis is dismal. To further improve EFS, the NOPHO-DB-SHIP consortium has initiated trial CHIP-AML22. Building on the successes achieved by the recent trial from this consortium (NOPHO-DBH AML-2012, preliminary data as per June 2023, n=878: 5-years pEFS 63%, 5-years pOS 78%), CHIP-AML22 will incorporate innovative elements, mainly the FLT3-inhibitor quizartinib (Vanflyta®) that was recently approved by the FDA and PMDA in combination with chemotherapy for the treatment of newly diagnosed adult AML patients with FLT3-ITD mutations, and gemtuzumab ozogamicin (GO, Mylotarg ®) that improved outcome in adult and pediatric AML when added to chemotherapy. CHIP-AML22 will maintain the approach of treatment-response driven risk-adapted treatment, intensified induction treatment for patients with ≥5% AML cells in the bone marrow (BM) after course 1, and consolidation with allogeneic stem cell transplantation (allo-SCT) for high-risk patients after the first consolidation course HAM. Study Design and Methods This clinical trial will enroll newly diagnosed de novo patients aged ≥1 day to ≤18 years to 2 randomized studies in the Master protocol, to the linked quizartinib trial, or the standard arm of the Master (Figure 1). Innovations include: (1) a phase II, single arm, open-label study on the safety, efficacy, pharmaco-kinetics and -dynamics of quizartinib in combination with chemotherapy, and as single-agent after allo-SCT, in FLT3-ITD/ NPM1wt AML; All patients will receive quizartinib monotherapy for 6 months post-SCT, except those that were negative for measurable residual disease determined with multiparameter flow cytometry (MFC-MRD) in BM1 and remained negative (study CHIP-AML22/Quizartinib); (2) a phase III, open-label randomization with gemtuzumab ozogamicin (GO; Mylotarg®) added to induction therapy MEC for pediatric CD33-positive newly diagnosed AML patients aimed at improved outcome (study Ri), (3) a phase III, open-label randomization with three (standard of care) versus two (investigational) courses of consolidation chemotherapy for standard-risk patients as non-inferiority trial (study Rc), and (4) refining risk-group adapted treatment, aimed at improved outcome. Novel high-risk categories are patients with RAM-phenotype and/or CBFA2T3::GLIS2, and patients with KMT2A-rearranged AML (excluding KMT2A::MLLT3) with ≥0.1% AML cells after course 1 in BM1. Patients with ≥15% AML cells on day 22 BM after course 1, or ≥0.1-5% AML cells by MFC-MRD after course 2 in BM2 (end-of-induction), or those with FLT3-ITD/NPM1wt remain to be high-risk, similar to AML-2012. Further, dexrazoxane is recommended to all patients before receiving daunorubicin or mitoxantrone, aiming to prevent late-onset cardiotoxicity. The quizartinib trial includes a safety run-in with a modified rolling-6 design and two dose levels with 6 evaluable patients per dose level. In study Ri, patients assigned to the experimental GO-arm will receive GO twice during induction with MEC at a dose of 3 mg/m 2 (max 5 mg) with an interim-analysis for superiority of any of these two arms. The investigational arm of the Rc randomization will omit HA 3E. Both the quizartinib trial and each randomization will also be compared to historical controls from the AML-2012 trial. CHIP-AML22 expects to enroll 130-140 patients per year and it will take 7-8 years to reach the total number of 905 patients needed to answer all study questions. Patients will be followed-up for at least 5 years from diagnosis and thus, the total study duration is expected to be 12-13 years. The Master protocol (EU CT: 2023-504999-25) and linked quizartinib trial (EU CT: 2023-505000-27-00) were approved by the Dutch Ethical Review Authority and as of July 2023, enrollment in the Master protocol including study Rc has started in The Netherlands. Study Ri and the linked quizartinib trial are expected to open in Q4 2023. At least 15 other countries with nearly 60 sites will participate in CHIP-AML22: Belgium, Denmark, Estonia, Finland, Hong Kong, Iceland, Israel, Latvia, Lithuania, Norway, Portugal, Spain, Sweden, Switzerland and Uruguay.

CLEC2A Is a Novel AML-Restricted Immunotherapeutic Target Enriched in KMT2A-Rearranged Acute Myeloid Leukemia

KMT2A rearrangements (KMT2A-r) are a frequent event in childhood acute myeloid leukemia (AML) and in treatment related AML in adults leading to a highly refractory and deadly subtype of AML regardless of age. Targeted therapies for KMT2A-r AML have had limited success with novel therapies urgently needed. Despite successes in other leukemias, immunotherapy in AML is in its infancy in part due to significant overlap of antigens expressed in AML and normal hematopoietic cells, which would limit efficacy and increase hematopoietic toxicity. We interrogated the AML transcriptome compared to normal hematopoiesis to identify optimal targets that are silent in normal hematopoieisis and are highly expressed in AML. Here we describe a novel immunotherapeutic target, CLEC2A, a member of the C-type lectin family, that is highly enriched in high-risk AML, particularly KMT2A-r AML and absent in normal hematopoeisis. We evaluated the association between KMT2A fusions and CLEC2A expression identifying CLEC2A as a putative oncoprotein and the potential for CLEC2A to serve as an immunotherapeutic target in high-risk AML via antibody-mediated cytotoxicity. Diagnostic specimens from 1,864 patients with AML (Pediatric 0-29 years from COG Phase III trials N=1486; Adult 18-88 years SWOG N=199 and TCGA-LAML N=179) were interrogated using contemporary NGS to identify genetic aberrations. Conventional karyotyping and mutational analysis were used to determine additional cytogenetic and molecular abnormalities. As CLEC2A was entirely absent in normal bone marrow (TPM 0-0.15), positive CLEC2A expression was defined as mRNA transcripts per million (TPM) &amp;gt;1. Although, CLEC2A was detected in only 18% of AML patients (252/1486; 17% pediatric and 90/378; 24% adult patients), it was highly enriched in those with KMT2A-r AML with 77% (N=193) of CLEC2A positive patients having KMT2A-r, accounting for 48% of the total KMT2A-r cohort, Fig 1A. CLEC2A expression was enriched in specific fusion subgroups: MLLT10 (80%), MLLT4 (71%), and MLLT3 (48%), Fig 1B. CLEC2A expression was low to absent in a majority of other patients, though was seen in other high-risk groups including non-KMT2A MLLT10 fusions, NUP98 fusions, CBFA2T3-GLIS2 fusions, and monosomy 7. CLEC2A is also expressed in a subtype of T-ALL (HOXA activated), which is a fusion driven T-ALL. We further evaluated a potential causal link between KMT2A fusions and CLEC2A expression and inquired whether KMT2A fusion proteins might directly bind to the CLEC2A promoter and induce CLEC2A expression. We used AutoCUT&amp;RUN profiling of genome-wide occupancy of KMT2A oncoproteins to evaluate direct binding of the KMT2A fusion protein to the CLEC2A promoter in primary AML patient samples with three different KMT2A fusions (MLLT10, MLLT4 and MLLT3). We found direct binding of KMT2A fusion proteins to the CLEC2A promoter region, indicating a causal relationship between KMT2A fusions and CLEC2A expression. We then evaluated the potential of therapeutic targeting of CLEC2A by an antibody-drug conjugate using a HumZap cytotoxicy assay. CLEC2A antibody conjugated to saporin (a toxin with cytolytic activity when internalized) was incubated with CLEC2A+ and CLEC2A- cells with target-specific cytotoxicity evaluated by colorimetric assay. CLEC2A+ OCI-AML2 cells had significantly higher rate of cytotoxicity compared to CLEC2A- control cells at 10nM Ab-toxin conjugate. Clinical outcomes for patients with and without CLEC2A expression was evaluated. Patients with CLEC2A had significantly worse outcomes (EFS 23% vs. 45.5%, p&amp;lt;0.0001 and OS 38% vs. 63%, p&amp;lt;0.0001 for patients with and without CLEC2A expression, respectively). Given high rates of co-occurring KMT2A-r AML, particularly high-risk fusions (MLLT10 and MLLT4), we evaluated clinical outcomes within the KMT2A-r cohort as these patients have poor EFS and OS. KMT2A-r patients with CLEC2A had significantly worse EFS (24% vs. 39%, p=0.0063) and OS (38% vs. 60%, p=0.0002). Within the KMT2A-r cohort, high rates of relapse were seen for patients with CLEC2A expression compared to those without CLEC2A (72% vs. 55%, p=0.0039). Here we describe CLEC2A, a novel AML-restricted cell surface target that is an ideal immunotherapeutic target. CLEC2A is highly expressed in KMT2A-r AML, entirely absent in normal hematopoietic cells, directly and causally linked to the KMT2A fusion, and can be used for target-directed cytotoxicity.

Targeting HDAC9 Contributes to Degradation of MLL Fusion Oncoproteins in KMT2A-Rearranged Acute Myeloid Leukemia

Acute myeloid leukemia with MLL rearrangement (MLLr-AML) is a highly aggressive subtype of AML. Existing treatment strategies are nonspecific and ineffective. The MLL fusion gene is one of the driver oncogenes in leukemia and is central to the pathogenesis of MLLr-AML, and more research has recently begun to focus on targeting MLL fusion proteins. Targeting MLL fusion proteins has been shown to have a tumor suppressor effect, demonstrating efficacy in preclinical models of MLLr-AML, and in 2022 Scott A Armstrong ‘s team suggested that degradation of MLL-AF9 can cause an increase in cell differentiation and apoptosis, confirming the efficacy of targeting MLL fusions. However, specific targets that degrade MLL fusion proteins as well as clinically translatable targeted therapies are still lacking and need to be further explored. Study confirmed that MLL-rearranged ALL cells are highly sensitive to the broad-spectrum HDAC inhibitor panobinostat. A recent clinical study by Mireia Camós’ team proposed that HDAC9 has a significantly high expression in childhood MLL-rearranged B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and AML, but that article did not address any mechanistic studies. The potential value of HDAC9 in adult lymphoid hematologic diseases has been gradually revealed, however, the expression profile and precise molecular mechanisms of HDAC9 in adult myeloid hematologic tumors, have not been reported, which deserves to be further explored. In order to investigate the expression and role of HDAC9 in MLLr-AML, we analyzed the second-generation sequencing data from the Beat AML dataset and found that HDAC9 has a significantly high expression in MLLr-AML, while only HDAC9 is characterized by this feature in HDAC family molecules. Further we analyzed the data of 300 adult AML specimens in our hospital (including 20 MLLr-AML patients), and the results further confirmed that only HDAC9 among the HDAC family had a significantly higher expression level in the MLLr-AML patient group than that in the Non-MLLr-AML patient group (P&amp;lt;0.001), and the high expression of HDAC9 in the MLLr-AML patient group was a common phenomenon and not limited to a particular class of MLL chaperone molecules. Further studies revealed that overexpression of MLL fusion proteins in HEK293T could cause up-regulation of HDAC9 protein levels, and this experiment clarified the direct correlation between the MLL fusion genes and the high expression of HDAC9. The HDAC IIa inhibitor (TMP-269), which can target HDAC9, was significantly more sensitive in MLLr-AML cell lines than in Non-MLLr-AML cell lines, and also that MLLr-AML cell survival was dependent on HDAC9, and that knockdown of HDAC9 induced an increase in apoptosis and a slowdown in proliferation in MLLr-AML cells. We subsequently demonstrated that knockdown of HDAC9 downregulates the expression of MLL fusion proteins through degradation of MLL proteins. Furthermore, when exploring therapeutic options for targeting MLLr-AML, we found that combining the BCL-2 inhibitor Venetoclax (VEN) with an MLL-Menin specific inhibitor (MEN1i) specifically downregulated the expression of HDAC9 and had a favorable synergistic killing effect on MLLr-AML in both in vitro and in vivo models, further suggesting an important role of HDAC9 in the treatment of MLLr-AML. To sum up, through this study, we can shed light on the role of specific HDAC molecules in MLLr-AML and provide a new potential target for the degradation of MLL fusion protein to eradicate MLLr-AML.

Revumenib Monotherapy in Patients with Relapsed/Refractory KMT2Ar Acute Leukemias: Efficacy and Safety Results from the Augment-101 Phase 1/2 Study

Background: Revumenib (SNDX-5613), a potent, selective small-molecule inhibitor of the menin-histone-lysine N-methyltransferase 2A (KMT2A) interaction, is being investigated in patients (pts) with relapsed/refractory (R/R) KMT2A-rearranged ( KMT2Ar) and nucleophosmin 1-mutated ( NPM1m) acute leukemias. An initial analysis of the phase 1 of AUGMENT-101 (NCT04065399) has been reported ( Nature. 2023;615:920-924). Here we report an update of &amp;gt;1 year additional experience in the phase 1 portion of the study, which has completed enrollment, with safety data from 131 treated pts and efficacy data in 80 pts with R/R KMT2Ar leukemia. Demographic data from pivotal phase 2 cohorts are included; safety and efficacy data of a preplannedanalysis will be reported at the meeting. Methods: In phase 1, pts aged ≥30 days with R/R acute leukemias were assigned to 1 of 6 dose-escalation arms designed to identify a recommended phase 2 dose (RP2D) for concomitant administration of weak, moderate, or strong cytochrome P450 3A4 inhibitor (CYP3A4i) azole antifungal or no CYP3A4i. Pts received revumenib every 12 hours (q12h) or 3 times a day at a flat dose (≥40 kg) or a body surface area-based dose (&amp;lt;40 kg). Safety was assessed in all pts who received ≥1 dose of revumenib; responses are reported for all treated pts with KMT2Ar. In pts who underwent hematopoietic stem cell transplant (HSCT), responses reported are those achieved before HSCT. The Phase 2 study was initiated following identification of an RP2D of 163 mg (95 mg/m 2 if &amp;lt;40 kg) q12h with a strong CYP3A4i in 28-day cycles until unacceptable toxicity, lack of at least morphological leukemia-free state (MLFS) by end of cycle 4, or progressive disease without clinical benefit as defined by the investigator. The phase 2 primary objectives are evaluation of safety and tolerability of revumenib and assessment of the complete remission (CR) + CR with partial hematologic recovery (CRh) rate. The safety and efficacy of revumenib at an RP2D is being explored in 3 expansion cohorts: Cohort 2A, KMT2Ar acute lymphoblastic leukemia (ALL)/mixed phenotype acute leukemia; Cohort 2B, KMT2Ar acute myeloid leukemia (AML); and Cohort 2C, NPM1m AML. A preplanned pooled analysis of adult and pediatric pts with KMT2Ar acute leukemia will be conducted when a subset of Cohorts 2A+2B pts have completed a minimum of 4 cycles or have discontinued therapy. Results: Phase 1: As of June 19, 2023, 80 pts with R/R KMT2Ar acute leukemia were treated with revumenib in the 6 dose-escalation arms (Table 1). In adults with AML (n=51), the largest KMT2Ar subgroup, CR+CRh was 28% and overall response rate (ORR) was 59%. Responses were consistent in ALL/other leukemia subtype (n=15), with 27% CR+CRh and 53% ORR. ORR (61%) was similar in pediatric pts (&amp;lt;18 years, n=18), with 22% proceeding to HSCT. Although similar percentages of adult and children proceeded to transplant, consistent with common clinical practice in pediatrics, 75% of children transplanted went on to HSCT prior to achieving a CR/CRh (2 MLFS, 1 CRp) versus 17% of adults. In the safety population (n=131), 24% of pts had a ≥Grade 3 treatment-related adverse event (TRAE), with Grade 3 QTc prolongation in 8% and differentiation syndrome in 2% (Table 1). QTc prolongation remained as the only dose-limiting toxicity; no patients experienced ventricular arrhythmias. In addition, based on updated phase 1 pharmacokinetics, clinical activity, and safety data, an RP2D of 276 mg q12h without a strong CYP3A4i was established. Phase 2: In a preliminary analysis of Cohorts 2A and 2B (June 19, 2023), 88 pts with KMT2Ar acute leukemia were enrolled and treated with revumenib at an RP2D (Table 2). Ages ranged from 1.3 to 75 years (22 aged &amp;lt;18 y; Table 2). Leukemia subtypes included ALL (15%), AML (81%), and other (3%). Pts were heavily pretreated, having received a median of 3 prior lines of therapy (range 1-12); 38% received ≥4 prior lines and 48% had prior HSCT. Conclusions: These phase 1 data continue to demonstrate deep responses to revumenib and a manageable safety profile in heavily pretreated patients with R/R KMT2Ar acute leukemia across ages and subtypes. The lack of targeted therapies approved for KMT2Ar leukemia indicate the need for further investigation of novel, potentially transformative therapies such as revumenib. Enrollment in the AUGMENT-101 trial is ongoing. Pivotal Phase 2 safety and efficacy data for the KMT2Ar population will be reported at the meeting.

Prognostic Role of Molecular MRD Variations during Treatment of Pediatric AML: A Retrospective AIEOP AML2013/01 Study

Background. Measurable residual disease (MRD) is a key parameter to evaluate response to cytotoxic treatment in leukemia patients and, thus, to guide therapeutic strategies. In fact, the identification of persisting leukemia cells after chemotherapy is associated with an increased risk of disease recurrence, and many patients with acute myeloid leukemia (AML) experiencing relapse have a fatal outcome. Many somatic genetic aberrations are under investigation for assessing molecular quantitative MRD (qMRD). However, while flow cytometry is already recognized as useful in measuring residual disease levels, the impact of qMRD monitoring is still debated. Methods and Results. We set up a qPCR assay to quantify mRNA fusion or mutated transcripts for 23 AML rearrangements identified at diagnosis in 247 patients (67% of the whole population) enrolled in our recently concluded AIEOP-AML2013/01 trial, with a sensitivity reaching 10 -5/-6. We measured qMRD after the induction therapy courses (n=202) and identified 66 cases with qMRD&amp;gt;1x10 -3 (cut-off determined by ROC curve method, qMRD with respect to disease levels found at disease onset) and 136 with qMRD&amp;lt;1x10 -3 after the II induction course. Patients with qMRD&amp;gt;1x10 -3 had a significantly worse OS and EFS with respect to patients with qMRD&amp;lt;1x10 -3 (77% vs 89%, p=0.016 and 61% vs 78%, p=0.016, respectively). qMRD assessment within each AML risk group, namely Standard (SR, n=64), Intermediate (IR, n=38) or High (HR, n=101), showed that the persistence of qMRD&amp;gt;1x10 -3 after the II induction course in SR patients did not correlated with worse EFS with respect to patients who reduced qMRD levels below the cut-off, mainly due to NPM1 mutated patients all relapsing (7/7) with qMRD&amp;lt;1x10 -3 after the second induction course. By contrast, qMRD&amp;gt;1x10 -3 was a predictive variable able to discriminate, among the patients assigned to the IR (EFS 46% vs 81% for IR, p=0.009) those children with poorer outcome. For the HR patients, qMRD 1x10 -3 predicted a worse, although not statistically significant, outcome (61% vs 80% for HR, p=0.08). When we interrogated qMRD role within the different genetic subtypes, we showed that qMRD significantly impacted on EFS of patients harboring t(8;21) RUNX1-RUNX1T1 (47% for qMRD&amp;gt;1x10 -3 vs 88% for qMRD&amp;lt;1x10 -3, p=0.004), this finding corroborating results previously published by our group. A similar trend, although not significant, was also obtained for patients harboring isolated FLT3-ITD mutation, who had a dismal EFS when qMRD levels after the II induction course were above 1x10 -3 (40% (n=5) vs 71% (n=7) for qMRD&amp;lt;1x10 -3, p=ns). qMRD reduction for KMT2A-rearranged AML cases and for other genetic markers having a lower incidence in AML, needs to be evaluated in larger cohorts to obtain robust results. Additionally, we interrogated if qMRD maintains the same clinical relevance when measured after the III or IV chemotherapy blocks. We calculated proper cut-off for this analysis by ROC curve method, finding again 1x10 -3 for the third chemotherapy cycle; we observed that AML cases with qMRD&amp;gt;1x10 -3 had a worse EFS with respect to patients with lower levels of qMRD (39% n=30 vs 80% n=82, p&amp;lt;0.0001). As concerns the IV chemotherapy block, we calculated an appropriate cut-off corresponding to 1x10 -4, and once again we confirmed that patients with qMRD above the cut-off displayed a lower probability of experiencing adverse events (EFS 49% n=46 vs 85% n=65, p=0.0003). In conclusion, this is the first study that considers a serial and continuous qMRD monitoring all along an entire trial demonstrating a significant prognostic role of qMRD after II, III and IV therapy courses. These findings demonstrate that qMRD represents a useful tool in supporting therapeutic decisions and suggest that harmonization of currently used molecular MRD detection methods is the best solution to face MRD challenge in pediatric AML. Further comparisons between molecular and cytofluorimetric methods will increase the chances to identify those patients at high risk of relapse, despite negative MRD measured by flow cytometry.

LAMP5 Is an AML-Restricted Immunotherapeutic Target Enriched in High-Risk Acute Myeloid Leukemia

LAMP5, Lysosomal Associated Membrane Protein Family Member 5, a neuronal protein that plays a role in synaptic plasticity is normally found primarily in the brain, however limited studies have identified it to be expressed in leukemia, particularly KMT2A-rearranged leukemias. Importantly, there is low expression of LAMP5 in normal hematopoiesis making it an ideal potential immunotherapeutic target for both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). As a newly identified aberrant protein in leukemias, there is little data available on the prevalence or clinical implications of LAMP5 expression. We interrogated the transcriptome and associated clinical and laboratory data from a large cohort of pediatric AML patients to define the prevalence, co-occurring cytomolecular aberrations and clinical outcomes associated with LAMP5 expression. Ribo-depleted RNA seq data from 2,241 patients with AML (Pediatric ages 0-29 years N=1,412, from recent Phase 3 COG clinical trials; Adult ages 18-88 years from BEAT AML N=451, SWOG N=199, TCGA-LAML N=179) with available cytogenetic, molecular, and clinical data were utilized. Gene fusions, internal tandem duplications (ITD) and partial tandem duplications (PTD) were detected by Cicero. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytogenetic and molecular abnormalities. Response was measured by 5-year overall survival (OS), event-free survival (EFS) and relapse risk (RR). Of the 1,412 pediatric patients, LAMP5 was expressed (positive defined as mRNA transcripts per million (TPM) &amp;gt;2) in 396 patients (28%) with mRNA TPM ranging from 0-884 with similar prevalence found in adult AML (28%; BEAT AML N=126/452, SWOG N= 59/199 TCGA N=50/179), but with significantly higher TPM ranging from 0-3,200. Of the 396 pediatric patients with LAMP5 expression, there was a notable enrichment in KMT2A-r AML (N=203, 51%), which accounts for 59% of the total KMT2A-r cohort. Within the KMT2A-r cohort, there is enrichment in the MLLT1 (N=17/18, 94%), MLLT3 (N=61/93, 66%) and MLLT10 (N=58/69, 84%) fusion groups. Additionally, LAMP5 expression was seen in other high risk fusion subtypes including 100% of patients with KAT6A-CREBBP (N=12/12) and 29% of NUP98 fusions (N=45/154). There is absent LAMP5 expression in normal lymphoid and myeloid cells, but there is moderate expression seen in plasmacytoid dendritic cells, which could provide a therapeutic target for Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN). Given the association with high-risk AML fusion subtypes, the impact of LAMP5 on clinical outcome in AML pediatric patients was evaluated. High rates of relapse were seen in patients with LAMP5 expression, with a relapse rate of 45% compared to 36% in LAMP5-negative patients. 5 year event-free survival (EFS) was 36% for LAMP5-positive patients compared 45% for LAMP5-negative patients (p=0.0005). Similar poor outcomes were seen with overall survival 54% for LAMP5-positive patients vs. 62% for LAMP5-negative patients (p=0.001). In an analysis of a large cohort of AML patients, we found that LAMP5 expression is highly enriched in many high-risk fusion subtypes including KMT2A-rearranged AML, KAT6A-CREBBP and NUP98 fusions. Patients with LAMP5 expression have high rates of relapse with significantly worse outcomes compared to patients without LAMP5. Given low levels of LAMP5 in normal hematopoiesis, LAMP5 is an ideal AML-restricted immunotherapy target with broad applicability for many high-risk fusion subtypes of AML. Current efforts to generate a LAMP5 chimeric antigen receptor (CAR) T cell are underway which will be used to evaluate preclinical efficacy in AML via cell- and patient-derived xenograft models with the goal to translate this novel therapy into clinical use to improve the current dismal outcomes for these high-risk patients.

Spatial Technologies Reveal the Immune Landscape of Pediatric Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) is a cancer with a particularly low mutational burden in comparison to other pediatric and adult cancers and therefore, thought to be a poor candidate for T cell-engaging immunotherapies (Gröbner et al., 2018; Pfister et al., 2022). However, little is known about the composition and function of T cells in the bone marrow (BM) of pediatric AML patients. Insight into the frequency and function of BM T cells in children with AML is relevant for naturally occurring AML immune defenses, as well as for T cell-engaging immunotherapies (Koedijk et al., 2021). Here, we performed a multidimensional characterization of the tumor immune microenvironment in children with newly diagnosed AML. We obtained 82 formalin-fixed and paraffin-embedded (FFPE) diagnostic bone biopsies from a representative cohort of pediatric patients with treatment-naïve de novo AML (N=72) and from age- and sex-matched pediatric patients with treatment-naïve early-stage rhabdomyosarcoma without malignant BM infiltration (non-leukemic controls, N=10). We employed immunohistochemistry (IHC) alongside immune-related gene expression profiling (NanoString PanCancer IO 360 TM Panel) and spatial transcriptomics (NanoString GeoMx TM Whole Transcriptome Atlas). Moreover, we acquired an additional dataset of pre-treatment immune-related gene expression profiling obtained from the BM of 30 flotetuzumab-treated (CD3 x CD123 bispecific antibody) refractory/relapsed (R/R) adult AML patients (NCT02152956; Vadakekolathu et al., 2020) for TIDE-based immune deconvolution (Jiang et al., 2018). Using IHC, we identified (a trend towards) a decreased abundance of the overall number of T cells ( P=0.09) and CD8 + T cells ( P=0.011; Panel A) in the BM of pediatric AML patients in comparison to non-leukemic controls, respectively. Notably, the extent of overall T cell and CD8 + T cell infiltration could differ up to 180-fold between individual AML patients. In general, this difference in T cell infiltration was not associated with the abundance of leukemic blasts or patient's cytogenetic alterations. Nevertheless, children with complex karyotype AML had higher numbers of BM T cells compared to children with normal karyotype AML ( P=0.03).Using differential gene expression analysis, we identified genes related to T cell-attracting chemokines, cytotoxicity, and immune checkpoints to be significantly upregulated in T cell-infiltrated- compared to T cell-depleted samples. Moreover, the ratio of anti- to pro-inflammatory macrophages (M2/M1-ratio) was significantly lower in T cell-infiltrated- compared to T cell-depleted pediatric AML samples ( P&amp;lt;0.001). Interestingly, this M2/M1-ratio was also significantly lower in R/R adults that responded to flotetuzumab immunotherapy in comparison to non-responders ( P&amp;lt;0.001). In fact, this M2/M1-ratio outperformed several other known predictors of response to flotetuzumab immunotherapy (AUROC: 0.852 (95% CI: 0.71-0.99), P=0.001; Jiang et al., 2018; Ayers et al., 2017). In addition, IHC revealed that 9 immune-infiltrated samples, including 6 KMT2A-rearranged AML samples, harbored large networks of T- and B cells (representative image of B cell-networks shown in panel B). Using spatial transcriptomics, we dissected the composition of these lymphoid aggregates and revealed localized anti-tumor immunity in the BM of AML. In comparison to tumor areas, these aggregates showed a higher abundance of activated cytotoxic T cells ( P&amp;lt;0.001), memory B cells ( P&amp;lt;0.001), and plasma cells ( P&amp;lt;0.001), suggesting the presence of tertiary lymphoid structure-like (TLS-like) aggregates in the BM of AML. In conclusion, we identified a subset of pediatric AML patients with relatively high T cell infiltration and a relatively low abundance of anti-inflammatory macrophages in the BM at diagnosis. Furthermore, we found that the BM M2/M1-ratio may be informative of response to T-cell engaging immunotherapies in adult AML, which needs to be validated in pediatric AML. Lastly, for the first time, we identified TLS-like aggregates in the BM of AML patients, which have been associated with immunotherapy response in many cancers (Schumacher &amp; Thommen, 2022). Additional studies to further characterize the function and relevance of these lymphoid aggregates are ongoing.

Drug Repurposing in a Zebrafish AML Model

Acute myeloid leukemia (AML) is a genetically heterogeneous hematological malignancy. AML arises from the stepwise accumulation of mutations in hematopoietic stem cells (HSCs). The t(9;11)(p22;q23) translocation is a translocation detected in about 5 to 7% of AML patients, which gives rise to the KMT2A/MLLT3 or MLL/AF9 ( MA9) fusion gene. Zebrafish (ZF) are well-established model organisms for investigating human hematopoiesis. Approximately 70% of human genes have corresponding orthologues in zebrafish, and over 80% of disease-related genes are conserved between the two species. Previously, we established a transgenic MA9 zebrafish AML model (Runx1+23:MA9), in which the AML was serially transplantable. Our investigations revealed that MA9 expression alters hematopoiesis in transgenic embryos before the onset of AML, leading to myeloid expansion. Whole-mount in situ hybridization (WISH) of embryos showed increased expression of granulocyte markers such as lyz 24 and 48 hours after fertilization (hpf). Based on these findings, we developed a Runx1+23:MA9/lyz-EGFP zebrafish leukemia line, in which lyz-expressing myeloid cells are EGFP positive. This model provides a phenotype-based platform to evaluate the therapeutic potential of any drug that can affect the myeloid cell population. We used this ZF model to investigate the therapeutic potential of nine drugs in MA9-driven AML. MA9 transgenic fish (Runx1+23:MA9) were crossed with lyz-EGFP fish to generate a MA9/lyz-EFGP line. In this line, myeloid cells can be visualized with a fluorescent microscope. The expression of lyz in the MA9/lyz-EFGP transgenic embryos was compared to that in the lyz-EGFP line. In our previous study on MA9 transgenic leukemic fish, RNA-seq analysis had revealed higher expression of genes acting in the EGFR, MEK, RAF, KRAS pathways and downregulation of genes in TP53. Based on these results, we selected nine drugs that are known to affect some of these pathways as well as drugs that are used to treat leukemia for screening in our model. Control and MA9/lyz-EFGP ZF 48 hpf embryos were treated with 10 µM of each drug. 24 hrs after drug treatment, the number of lyz expressing cells was analyzed by visualizing the embryos under a fluorescent microscope, and lyz transcript levels were measured by qPCR. Treatment with erlotinib, lapatinib, cytarabine, and gefitinib led to a visible reduction in the number of lyz-expressing cells in the MA9 transgenic embryos but not in the controls (Figure 1). In comparison, treatment with decitabine resulted in a decrease in the number of lyz expressing cells in both the MA9/lyz-EGFP and the control embryos. Treatment with doxorubicin, zoledronic and arsenic trioxide did not have an effect on the number of lyz-expressing population in the transgenic nor in the control embryos. These results were confirmed by qPCR, which showed that expression of lyz was reduced by 33% to 51% in the erlotinib, lapatinib, cytarabine, and gefitinib treated MA9 transgenic embryos (Figure 2). Treatment with these drugs did not significantly change the lyz expression in lyz-EFGP control embryos. Our results provide strong evidence for the successful establishment of a zebrafish model of KMT2A-rearranged AML that can be used for low to medium-throughput drug screening by employing the expression level of an endogenous gene (lyz) as a read-out. This model is a valuable tool to assess the drug's specificity in targeting oncogene-expressing cells and normal hematopoietic cells and, therefore, to identify novel therapeutic agents that can target the cancer cells with fewer side effects on normal hematopoietic cells.

Children's Oncology Group's 2023 blueprint for research: Myeloid neoplasms.

During the past decade, the outcomes of pediatric patients with acute myeloid leukemia (AML) have plateaued with 5-year event-free survival (EFS) and overall survival (OS) of approximately 46 and 64%, respectively. Outcomes are particularly poor for those children with high-risk disease, who have 5-year OS of 46%. Substantial survival improvements have been observed for a subset of patients treated with targeted therapies. Specifically, children with KMT2A-rearranged AML and/or FLT3 internal tandem duplication (FLT3-ITD) mutations benefitted from the addition of gemtuzumab ozogamicin, an anti-CD33 antibody-drug conjugate, in the AAML0531 clinical trial (NCT00372593). Sorafenib also improved response and survival in children with FLT3-ITD AML in the AAML1031 clinical trial (NCT01371981). Advances in characterization of prognostic cytomolecular events have helped to identify patients at highest risk of relapse and facilitated allocation to consolidative hematopoietic stem cell transplant (HSCT) in first remission. Some patients clearly have improved survival with HSCT, although the benefit is largely unknown for most patients. Finally, data-driven refinements in supportive care recommendations continue to evolve with meaningful and measurable reductions in toxicity and improvements in EFS and OS. As advances in application of targeted therapies, risk stratification, and improved supportive care measures are incorporated into current trials and become standard-of-care, there is every expectation that we will see improved survival with a reduction in toxic morbidity and mortality. The research agenda of the Children's Oncology Group's Myeloid Diseases Committee continues to build upon experience and outcomes with an overarching goal of curing more children with AML.

Palbociclib in Acute Leukemias With KMT2A-rearrangement: Results of AMLSG 23-14 Trial

Palbociclib in Acute Leukemias With KMT2A-rearrangement: Results of AMLSG 23-14 Trial

Measurable Residual Disease and Fusion Partner Independently Predict Survival and Relapse Risk in Childhood KMT2A-Rearranged Acute Myeloid Leukemia: A Study by the International Berlin-Frankfurt-Münster Study Group.

A previous study by the International Berlin-Frankfurt-Münster Study Group (I-BFM-SG) on childhood KMT2A-rearranged (KMT2A-r) AML demonstrated the prognostic value of the fusion partner. This I-BFM-SG study investigated the value of flow cytometry-based measurable residual disease (flow-MRD) and evaluated the benefit of allogeneic stem-cell transplantation (allo-SCT) in first complete remission (CR1) in this disease. A total of 1,130 children with KMT2A-r AML, diagnosed between January 2005 and December 2016, were assigned to high-risk (n = 402; 35.6%) or non-high-risk (n = 728; 64.4%) fusion partner-based groups. Flow-MRD levels at both end of induction 1 (EOI1) and 2 (EOI2) were available for 456 patients and were considered negative (<0.1%) or positive (≥0.1%). End points were 5-year event-free survival (EFS), cumulative incidence of relapse (CIR), and overall survival (OS). The high-risk group had inferior EFS (30.3% high risk v 54.0% non-high risk; P < .0001), CIR (59.7% v 35.2%; P < .0001), and OS (49.2% v 70.5%; P < .0001). EOI2 MRD negativity was associated with superior EFS (n = 413; 47.6% MRD negativity v n = 43; 16.3% MRD positivity; P < .0001) and OS (n = 413; 66.0% v n = 43; 27.9%; P < .0001), and showed a trend toward lower CIR (n = 392; 46.1% v n = 26; 65.4%; P = .016). Similar results were obtained for patients with EOI2 MRD negativity within both risk groups, except that within the non-high-risk group, CIR was comparable with that of patients with EOI2 MRD positivity. Allo-SCT in CR1 only reduced CIR (hazard ratio, 0.5 [95% CI, 0.4 to 0.8]; P = .00096) within the high-risk group but did not improve OS. In multivariable analyses, EOI2 MRD positivity and high-risk group were independently associated with inferior EFS, CIR, and OS. EOI2 flow-MRD is an independent prognostic factor and should be included as risk stratification factor in childhood KMT2A-r AML. Treatment approaches other than allo-SCT in CR1 are needed to improve prognosis.