Human AML Research Articles

The small inhibitor WM-1119 effectively targets KAT6A-rearranged AML, but not KMT2A-rearranged AML, despite shared KAT6 genetic dependency

BackgroundThe epigenetic factors KAT6A (MOZ/MYST3) and KMT2A (MLL/MLL1) interact in normal hematopoiesis to regulate progenitors’ self-renewal. Both proteins are recurrently translocated in AML, leading to impairment of critical differentiation pathways in these malignant cells. We evaluated the potential of different KAT6A therapeutic targeting strategies to alter the growth of KAT6A and KMT2A rearranged AMLs.MethodsWe investigated the action and potential mechanisms of the first-in-class KAT6A inhibitor, WM-1119 in KAT6A and KMT2A rearranged (KAT6Ar and KMT2Ar) AML using cellular (flow cytometry, colony assays, cell growth) and molecular (shRNA knock-down, CRISPR knock-out, bulk and single-cell RNA-seq, ChIP-seq) assays. We also used two novel genetic murine KAT6A models combined with the most common KMT2Ar AML, KMT2A::MLLT3 AML. In these murine models, the catalytic activity of KAT6A, or the whole protein, can be conditionally abrogated or deleted. These models allowed us to compare the effects of specific KAT6A KAT activity inhibition with the complete deletion of the whole protein. Finally, we also tested these therapeutic approaches on human AML cell lines and primary patient AMLs.ResultsWe found that WM-1119 completely abrogated the proliferative and clonogenic potential of KAT6Ar cells in vitro. WM-1119 treatment was associated with a dramatic increase in myeloid differentiation program. The treatment also decreased stemness and leukemia pathways at the transcriptome level and led to loss of binding of the fusion protein at critical regulators of these pathways. In contrast, our pharmacologic and genetic results indicate that the catalytic activity of KAT6A plays a more limited role in KMT2Ar leukemogenicity, while targeting the whole KAT6A protein dramatically affects leukemic potential in murine KMT2A::MLLT3 AML.ConclusionOur study indicates that inhibiting KAT6A KAT activity holds compelling promise for KAT6Ar AML patients. In contrast, targeted degradation of KAT6A, and not just its catalytic activity, may represent a more appropriate therapeutic approach for KMT2Ar AMLs.

Targeting the membrane-proximal C2-set domain of CD33 for improved CAR T cell therapy

Current CD33-targeted immunotherapies typically recognize the membrane-distal V-set domain of CD33. Here, we show that decreasing the distance between Tcell and leukemia cell membrane increases the efficacy of CD33 chimeric antigen receptor (CAR) Tcells. We therefore generated and optimized second-generation CAR constructs containing single-chain variable fragments from antibodies raised against the membrane-proximal C2-set domain, which bind CD33 regardless of whether the V-set domain is present (CD33PAN antibodies). CD33PAN CAR Tcells resulted in efficient tumor clearance and improved survival of immunodeficient mice bearing human AML cell xenografts and, in an AML model with limited CD33 expression, forced escape of CD33neg leukemia. Compared to CD33V-set CAR Tcells, CD33PAN CAR Tcells showed greater invitro and invivo efficacy against several human AML cell lines withdiffering levels of CD33 without increased expression of exhaustion markers. CD33PAN moieties were detected at a higher frequency on human leukemic stem cells, and CD33PAN CAR Tcells had greater invitro efficacy against primary human AML cells. Together, our studies demonstrate improved efficacy with CAR Tcells binding CD33 close to the cell membrane, providing the rationale to investigate CD33PAN CAR Tcells further toward possible clinical application.

PHF6 suppresses self-renewal of leukemic stem cells in AML

Acute myeloid leukemia is characterized by uncontrolled proliferation of self-renewing myeloid progenitors accompanied by a differentiation arrest. PHF6 is a chromatin-binding protein mutated in myeloid leukemias, and its isolated loss increases mouse HSC self-renewal without malignant transformation. We report here that Phf6 knockout increases the aggressiveness of Hoxa9-driven AML over serial transplantation, and increases the frequency of leukemia initiating cells. We define the in vivo hierarchy of Hoxa9-driven AML and identify a population that we term the “LIC-e” (leukemia initiating cells enriched) population. We find that Phf6 loss expands the LIC-e population and skews its transcriptome to a more stem-like state; concordant transcriptome shifts are also observed on PHF6 knockout in a human AML cell line and in PHF6 mutant patient samples from the BEAT AML dataset. We demonstrate that LIC-e accumulation in Phf6 knockout AML occurs not due to effects on cell cycle or apoptosis, but due to an increase in the fraction of its progeny that retain LIC-e identity. Our work indicates that Phf6 loss increases AML self-renewal through context-specific effects on leukemia stem cells.

Rapamycin increases leukemia cell sensitivity to chemotherapy by regulating mTORC1 pathway-mediated apoptosis and autophagy.

This study investigated the effect of rapamycin alone and in combination with chemotherapy (doxorubicin and cytarabine) on AML. Human acute monocytic leukemia cell line SHI-1 and NPG AML model mice created by intravenous injection of SHI-1 cell were treated with rapamycin, chemotherapy, or rapamycin plus chemotherapy. Analysis by cell counting kit-8, western blot, flow cytometry, and immunohistochemistry was performed, and results suggested that both rapamycin and chemotherapy inhibited proliferation of SHI-1 cells both in vitro and in vivo, suppressed neoplasm growth in vivo, and promoted survival of NPG AML mice. The antitumor effect of rapamycin plus chemotherapy was better than that of rapamycin alone and chemotherapy alone. In addition, western blot results demonstrated that rapamycin inhibited the phosphorylation of mTOR downstream targets 4EBP1 and S6K1 in SHI-1 cells, and increased the pro-apoptosis-related protein Bax and autophagy-associated proteins Beclin-1, LC3B-II, and ATG5 while reducing the anti-apoptosis-related protein Bcl-2. In conclusion, the results of this study indicate that rapamycin acts synergistically with doxorubicin and cytarabine in AML treatment, and its underlying mechanism might be associated with mTORC1 pathway-mediated apoptosis and autophagy.

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.

Sgr-2921, a Potent CDC7 Inhibitor, Demonstrates Significant Anti-Leukemic Responses in Patient-Derived AML Models Representing Difficult-to-Treat Disease

Introduction: Elevated replication stress (RS) is a common feature of many highly proliferative cancers including AML. CDC7 has been shown to activate key components of the RS response, including ATR, and the BRCA1 and cohesin complexes, thereby mitigating accumulation of DNA damage and genomic instability. Inhibition of CDC7 in cancer cells results in an impaired response to RS, leading to accumulation of DNA damage and cell death. SGR-2921 is a potent inhibitor of CDC7 and a clinical trial to evaluate the safety, tolerability and preliminary antitumor activity of SGR-2921 in patients with AML and MDS is expected to begin in 2023 (NCT 05961839). We have previously shown that SGR-2921 has potent anti-proliferative activity in 12 out of 16 patient-derived AML samples ex vivo. Importantly, 6 out of 6 AML patient samples with mutations in TP53 responded to SGR-2921. Here we show that SGR-2921 results in potent, dose-dependent, anti-leukemic activity in multiple disseminated in vivo AML patient-derived xenograft (PDX) models representing difficult-to-treat disease. Methods: NOG-EXL mice were inoculated with primary human AML blast cells after sublethal irradiation. Engraftment of human AML blast cells were measured in surrogate mice by flow cytometry using human CD45 vs. mouse CD45 markers. When human CD45 engraftment reached >20% of bone marrow cells in surrogate mice, the study mice were randomized based on weight and dosed with vehicle, SGR-2921 at 3 dose levels or a standard of care agent. SGR-2921 was administered via oral gavage (PO) twice daily in cycles of 5 days of dosing followed by 9 days of no compound dosing over 2 cycles. In a separate study, phosphorylated MCM2 (a direct substrate of CDC7) was measured in the same cohort of mice engrafted with human AML cells following 3 doses of SGR-2921 to determine target engagement. Results: In vivo, SGR-2921 showed strong anti-leukemic activity in the AML PDX models at tolerated doses. Specifically, engrafted AML cells derived from a patient with TP53 mutated AML, a relapsed and refractory patient and a patient with KTM2a rearrangements showed significant and dose-dependent reduction in human CD45 positive AML blast cells. The reduction of blast cells was observed in both peripheral blood and bone marrow. The reduction in human CD45 positive blast cells were associated with an increase in mouse CD45 positive cells. In addition, SGR-2921 demonstrated dose-dependent inhibition of phosphorylated MCM2 in the spinal cord and spleen of the AML PDX models as surrogates of the bone marrow and extravascular compartments, respectively. Conclusions: Our data show remarkable dose-dependent in vivo activity of SGR-2921 in AML PDX models, including in those representing difficult-to-treat disease. Direct inhibition of CDC7 by SGR-2921 in AML blasts was demonstrated by a dose-dependent reduction of phosphorylated MCM2. Together, these data demonstrate that SGR-2921-mediated CDC7 inhibition is an attractive novel treatment opportunity in AML, with potential utility in patients with high risk mutations and relapsed and refractory AML.

Forward Genetic Screen Implicates Drivers of Leukemic Progression in a Novel Model of Trp53 R270H myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is characterized by bone marrow failure and a highly variable clinical course. The most catastrophic complication of MDS is transformation to secondary acute myeloid leukemia (sAML). Notably, mutations in TP53 confer the single highest risk of transformation to sAML and death. However, some patients with TP53 mutated MDS do not develop sAML, suggesting that additional genetic events cooperate with TP53 mutations to transform MDS to sAML. Understanding the mechanisms of transformation of MDS to sAML could provide targets for therapeutic intervention. To model the genetics of MDS, we crossed mice bearing Trp53 R270H ( Trp53 is the murine TP53 gene) and deletion of genes syntenic with human chromosome 5q (del(5q)). To discover how additional mutations contribute to disease progression, we utilized Sleeping Beauty (SB) transposon mutagenesis in Trp53 R270H/del(5q) mice. SB transposase mobilized SB mutagenic T2/Onc transposons which randomly insert within the genome. T2/Onc transposons are designed to induce gain or loss of function alterations depending on the site and orientation of insertion with respect to targeted genes. We used the Mx1-Cre transgene to activate SB transposase and T2/Onc transposition in hematopoietic progenitors. Trp53 R270H anddel(5q)(or cytogenetically normal, CN) mice were crossed to SB mice to generate donor mice of the following genotypes: Trp53 R270H/del(5q)/ SB, Trp53 R270H/CN /SB, Trp53 WT/del(5q)/ SB, Trp53 WT/CN/ SB mice, and mice without SB transposition, (no transposition, NT: Trp53 R270H/del(5q) /NT). Bone marrow cells were transplanted into recipients, and SB insertional mutagenesis was activated using pI-pC to activate Cre. Mice receiving Trp53 WT/CN/ SB bone marrow developed more frequent T-cell leukemia (n=3/10) than myeloid leukemia (n=1/10). In contrast, mice receiving Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB bone marrow developed predominantly myeloid leukemia (n=14/28) more commonly than T-cell leukemia (1/28). Mixed phenotype leukemia was seen in 7/28 of these mice. Together, these data demonstrate a strong bias towards myeloid disease in SB-mutagenized Trp53 R270H bone marrow. To identify genes with SB insertions, we performed RNA sequencing to detect SB T2/Onc transposon-endogenous genefusion transcripts. Among Trp53 WT/CN/ SB leukemias, the most common recurrent SB fusions involved Notch1 and Ikzf1 as has previously reported for SB-associated T-cell leukemias . Among Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB leukemias, the most common recurrent SB-fusions involved Erg, Eras and Il2rb with Erg fusions detected 85% of Trp53 R270H leukemias (n=17/20). SB inserted upstream of Erg promoter indicating that these fusions likely upregulate expression of Erg. Indeed, Erg levels are significantly higher in leukemias that express SB-Erg fusions relative to leukemias that do not (p<0.0023). ERG is not recurrently mutated in human AML, but the ERG gene locus is commonly amplified, especially TP53 mutant AML. ERG is known to support normal hematopoietic stem cell self-renewal. Notably, Erg-insertions were also detected in a model of MDS expressing stabilized cyclin E with SB-mediated progression to erythroleukemia (Loeb 2019). Using gene set enrichment analysis, we found that hematopoietic stem cell and leukemic stem cell signatures are enriched in Erg-SB fusion leukemias. In our analyses of two independent data sets (TCGA and BEAT AML), stem cell signatures are also among the most highly enriched pathways in human AMLs expressing high ERG levels. Furthermore, in a human AML single cell RNA sequencing dataset (van Galen 2019), we found that ERG expression is highest in AML cells with the most immature stem and progenitor-like features. Together, these findings implicate a role for ERG as a driver of progression of MDS to AML by enhancing aberrant self-renewal. In summary, we present a novel murine model of Trp53/del(5q) MDS. In this model, Erg upregulation is associated with progression to AML and upregulation of leukemia stem cell gene expression profiles. These data implicate ERG as a major contributor to progression of MDS to secondary AML in the setting of mutant p53. Understanding the mechanisms of disease progression and self-renewal in myeloid malignancies with p53 mutations is critical to define effective therapeutic strategies in these rapidly fatal, treatment resistant diseases.

Gene Correction of DNMT3A:R882H in Primary Human AML Demonstrates That This Mutation Is Not Required for Disease Maintenance, but Is Associated with Increased Leukemia Stem Cell Frequency

Introduction: The genetic landscape of AML comprises numerous genetic alterations contributing to disease initiation and progression. Among these lesions, frequently occurring missense variants in DNMT3A:R882 are typically found as pre-leukemic events. An important and understudied question in leukemia pathogenesis is whether individual mutations are required for disease initiation, maintenance of established disease, or both. This is particularly the case with pre-leukemic mutations that affect the epigenome, as such mutations might be required to provide the epigenetic background necessary for disease initiation, but may no longer be required for disease maintenance. Here, we dissect the contribution of DNMT3A mutations to leukemic initiation and maintenance by gene correction in primary patient AML specimens. Methods: To functionally interrogate the contribution of DNMT3A mutations in human AML pathogenesis, we generated a novel CRISPR/Cas9-mediated toolset to selectively and specifically revert DNMT3A:R882 missense variants back to wildtype, or re-introduce the original mutation as a control, directly in both primary pre-leukemic HSCs (pHSCs) and primary leukemic blasts from 7 patient samples, leaving the remaining mutations and underlying germline background intact. Gene-corrected or re-mutated cells were subjected to functional assays both in vitro and in vivo. Results: We demonstrate that correction of DNMT3A mutations is feasible, achieving 5-40% gene editing both in purified pHSCs and leukemic blasts (n=7 patient samples). Using this approach, we first establish that DNMT3A mutations confer increased stemness in human pHSCs, with increasing serial replating capacity (mean increase re-mutated vs. corrected 5.53-fold, p<0.05). Surprisingly however, correction of DNMT3A mutations in leukemic blasts led to no immediate functional consequences. Specifically, primary patient leukemic blasts corrected for DNMT3A mutations retained the ability to engraft leukemia in NSGS mice and showed no difference in human chimerism or immunophenotype compared to re-mutated control from 5/5 primary patient samples. Importantly, limiting dilution transplantation assays (LDA) with two cases revealed no impact of DNMT3A mutations on leukemia-initiating cell (LIC) frequency in primary recipients, highlighting that DNMT3A mutations have no immediate effect on disease maintenance. We then performed whole-genome bisulfite sequencing and RNA-sequencing on leukemic blasts isolated from engrafted mice 4 months after DNMT3A correction or re-mutation. Global methylation levels between corrected and re-mutated cells were within the variance in either of these groups, and gene expression profiles were similarly driven by patient. However, paired analysis of each patient sample revealed that DNMT3A correction led to a consistent loss of stem cell expression signatures, suggesting influence of DNMT3A mutations on leukemia stem cell programs. Thus, we interrogated the effect of DNMT3A mutations on LSCs by performing secondary transplantations with two cases. In LDA assays, we observed a significant decrease in LIC frequency in the corrected leukemic blasts compared to re-mutated controls, indicating that correction of DNMT3A mutations induces a reduction in leukemia stem cell frequency, but does not completely abrogate leukemogenic potential. Conclusion: In summary, our work demonstrates a novel CRISPR/Cas9 approach to dissect the contribution of individual somatic lesions in human leukemia. The precise correction of single lesions while leaving co-occurring mutations intact allows us to perform controlled, functional genetic experiments on primary human AML. Here, we employ this approach to dissect the contribution of DNMT3A mutations to leukemia initiation and maintenance. We find that DNMT3A mutations impart increased stemness on human pHSCs, consistent with a key role in leukemia initiation. However, in primary patient blasts, we find that DNMT3A mutations are not required for leukemia maintenance. Strikingly, DNMT3A mutations in established leukemia were found to increase leukemia stem cell frequency. This approach will now allow for a detailed evaluation of differential methylation in mean and entropy and can be extended to other mutations of interest, extending our toolkit to unravel human AML biology directly in primary cancer specimens.

Evaluation of ICT01, a γ9δ2 T Cell-Activating Monoclonal Antibody, Combined with Venetoclax and Azacitidine in 1L AML (EVICTION Study)

Background: γ9δ2 T cells are emerging as a novel tumor-infiltrating effector cell that harbors strong cytolytic and pro-inflammatory activities, and whose intratumoral presence is associated with a favorable prognosis across solid and liquid cancer patients (Gentles et al., 2015; Tosolini et al., 2017). ICT01, a first-in-class anti-BTN3AmAb activating γ9δ2 T cells, completed Phase 1 testing in relapsed/refractory (r/r) solid tumors as monotherapy and in combination with pembrolizumab, and as monotherapy in r/r AML and lymphoma (EVICTION-NCT04243499). ICT01 activates circulating γ9δ2 T, CD8 T, and NK cells that leads to tumor infiltration and remodeling of the TME (De Gassart et al., 2021; Wermke et al., 2021) without any dose-limiting toxicities and a good safety profile. Encouraging clinical activity was observed in the hematologic cancer cohort with a 30% DCR on the 10 patients evaluable at week 8 or beyond (Garciaz, SITC 2023) supporting further clinical testing of ICT01 in combination with Venetoclax (VEN) and 5-Azacytidine (AZA), considered the standard of care for patients with newly diagnosed AML who are not candidates for intensive chemotherapy. Both ICT01 + γ9δ2 T cells and VEN/AZA induce killing of AML blasts, while VEN/AZA has been shown to enhance T-cell- and NK-cell-mediated cytotoxicity against AML blasts, suggesting that the combination of ICT01 plus VEN/AZA could lead to greater efficacy. We used preclinical models to demonstrate the ability of VEN/AZA to potentiate the anti-leukemic activity of ICT01-activated γ9δ2 T cells to support the clinical evaluation of ICT01 + VEN/AZA in 1L AML patients eligible to receive VEN/AZA. Results: Preclinical in vitro studies showed that ICT01-mediated activation of g9d2 T cells significantly increased survival against VEN toxicity by >50%, as compared to untreated control (p<0.01). Furthermore, killing assays performed by co-culture of AML blasts and HD-PBMC demonstrated that VEN/AZA enhances the blast killing capacity of ICT01-activated g9d2 T cells. These results were confirmed in vivo in NSG mice engrafted with human AML that were adoptively transferred with human g9d2 T cells and treated with VEN (40mg/kg/day OG)/AZA (2mg/kg/day IP), ICT01 (1mg/kg IV) or the combination. In this model the use of ICT01 together with VEN/AZA significantly delayed tumor growth (p<0.001) and significantly prolonged median survival to 42.5 days (vs 28 and 32.5 days for animals receiving ICT01 or VEN/AZA, respectively) (Figure 1). These results led to the design of the recently initiated Phase 2a arm of EVICTION in 1L AML where ICT01 is given at 10 mg or 75 mg IV Q4W on Day 1 of the 28-day approved regimen of VEN/AZA until toxicity or disease progression. Up to 25 patients will be treated at each dose level, following an interim analysis after 10 pts are treated at each dose. Clinical sites in France, Spain and the USA are participating. Biomarkers include multiparametric flow cytometry analysis of g9d2 T cells and other immune cells, and BTN3A expression in peripheral blood and in bone marrow aspirates at baseline and on Day 21-28. Circulating cytokine levels (including IFNγ, TNFa, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17a and MCP-1) are also assessed with MSD technology. Clinical response is evaluated per AML-specific standards (e.g., Cheson/IWG Criteria), with a target CR rate of >40%. Preliminary safety, biomarker and efficacy data from patients will be presented. Conclusion: Encouraging results obtained in the EVICTION Phase 1 trial and preclinical demonstration of the benefit of using ICT01 plus VEN/AZA allowed the initiation of a Phase 2a expansion cohort to evaluate the clinical benefit of this combination in 1L AML patients.

Immunosuppression in Isocitrate Dehydrogenase Mutated Acute Myeloid Leukemia

Introduction: Mutations in the isocitrate dehydrogenase (IDHmut) enzymes 1 and 2 are prevalent in a diverse array of human malignancies, including melanoma, glioma, acute immunoblastic T cell lymphoma (AITL) and acute myeloid leukemia (AML,15-20% of patients). IDHmut enzymes produce R-2-hydroxyglutarate (R-2-HG), an oncometabolite with immunomodulatory capability (Notarangelo et al, Science 2022). Immunotherapies in AML outside of allogeneic stem cell transplantation have proven to be largely ineffective, possibly due to immune evasion mechanisms intrinsic to myeloid malignancies. Herein, we aim to characterize the immune repertoire in IDHmut AML patients at the time of diagnosis, as well as the mechanisms by which R-2-HG may suppress immune cell function. Methods: The immune repertoire in cryopreserved peripheral blood mononuclear cells (PBMCs, n=10) and bone marrow mononuclear cells (BMMCs, n=8) of IDHmut AML patients were characterized using full spectrum flow cytometry to evaluate 30 different immune cell markers. T cell activation (CD25, CD69, CD40L, CD134), exhaustion (PD-1, CTLA-4, Lag-3, Tim-3), differentiation (Foxp3, CXCR3, CCR4, CCR6), and memory (CD45RA, CCR7, CD28, CD95) markers were assessed. IDH wild-type (IDHwt) AML PBMCs (n=4), BMMCs (n=12) and healthy volunteer PBMCs (n=6) and BMMCs (n=3) were used as controls. To investigate the effects of R-2-HG on CD4 + T cell activation, primary human naïve CD4 + T cells (CD4 +, CD45RA +) were isolated from healthy donors and activated (anti-CD3/CD28) in the presence of R-2-HG (20 mM) or PBS control. Activation, proliferation and cytokine production were assessed with flow cytometry, intracellular cytokine staining (ICS) and LEGENDplex at 4, 8, 24 and 72 hrs. The OT-II transgenic mouse model was used to assess antigen-specific T cell activation. T cell metabolism at rest and after activation in the presence of R-2-HG was evaluated using SCENITH (Argüello et al, Cell Metab 2021) and 13C 6-glucose isotope labeling coupled with mass spectrometry (MS) performed by the UCLA Metabolomics Center. Results: Full-spectrum flow cytometry analysis revealed a trend for higher CD4:CD8T cell ratio in AML BMMCs (fold=1.95, p=0.229) as well as increased CD4 + T cell activation and exhaustion markers compared to healthy donor BMMCs. No appreciable differences in CD4 + T cell markers were found between IDHwt AML and IDHmut AML, likely due to AML heterogeneity and small sample size. Nevertheless, our preliminary results suggest CD4 + T cell engagement in AML; thus, we chose to investigate how CD4 + T cells are affected by R-2-HG. Primary human naïve CD4 + T cells activated in the presence of R-2-HG exhibited a defect in early activation demonstrated by decreased CD69 expression (n=7, fold=0.773, p=0.001) and concomitant decreased production of IL-2 measured by ICS (n=7, fold=0.403, p=0.0001) and LEGENDplex (n=3, fold=0.557, p=0.046). Proliferation in the presence of R-2-HG was inhibited in a dose dependent manner (n=6, Division Index fold change=0.768, p=0.006 at 20mM). Antigen-specific OT-II CD4 + T cells showed a decrease in proliferation in response to OVA 323-339 (n=3, Division Index fold change=0.620, p=0.004). Given R-2-HG's structural similarity to α-ketoglutarate (αKG), we hypothesized that disruption in CD4 + T cell activation when cultured with R-2-HG may be due to metabolic dysfunction. Indeed, we found a decrease in global metabolism in CD4 + T cells cultured with R-2-HG (fold = 0.678, p = 0.009). MS revealed decreased levels of succinate and malate downstream and accumulation of αKG upstream of α-ketoglutarate dehydrogenase (KGDH) in the tricyclic acid (TCA) cycle, suggesting a defect in TCA flux (fold succinate / αKG ratio =0.134 unactivated, p = 0.029; 0.231 4h activation, p = 0.012; 0.328 24h activation, p = 0.004; Figure 1). Moreover, MS also showed increased amounts of essential amino acids (Ile, Leu, Met, Phe, Thr, Trp, Val, His; Figure 2), suggesting a compensatory reliance on amino acid import and metabolism. Conclusions: By characterizing primary AML samples at diagnosis, we found trending increases in CD4:CD8 ratio and expression of CD4 + T cell activation and exhaustion markers compared to healthy controls. We also demonstrate R-2-HG suppression of CD4 + T cells, potentially via inhibition of KGDH in the TCA cycle. Our studies show R-2-HG in IDHmut tumors can modulate CD4 + T cell function and contribute to immune escape in human AML.

Inactivation of DNA Polymerase Theta (PolΘ) Is Synthetic Lethal in DNMT3A Mutated Myeloid Malignancies - Potential Clinical Applications

Somatic mutations in DNMT3A are associated with unfavorable outcome in patients with AML, MPN and CML. DNMT3A mutations promote resistance to anthracyclines (including daunorubicin, the component of standard “7+3” induction therapy), interferon alpha, and ABL1 kinase inhibitor imatinib. Thus, malignant clones carrying DNMT3A mutations may be difficult to eliminate using standard treatments. AML, MPN and CML cells harbor oncogenic tyrosine kinase (OTK) such as FLT3(ITD), JAK2(V617F) and BCR-ABL1, respectively. We reported before that elevated levels of formaldehyde generated by altered serine/one-carbon cycle metabolism contributed to accumulation of highly lethal DNA double-strand breaks (DSBs) in OTK-positive cells. To protect malignant cells from DSB-induced apoptosis, OTKs regulate DNA damage response (DDR) mechanisms involving DSBs sensing (ATM and ATR kinases) and repairing (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, Polθ-dependent microhomology-mediated end-joining = TMEJ) as well as these activating cell cycle checkpoints (CHK1 and CHK2 kinases). Unfortunately, DNMT3A mutations caused resistance of OTK-positive cells to numerous DDR inhibitors (DDRis). DDRis sensitivity screen and synthetic lethal CRISPR/Cas9 screen revealed that OTK-positive cells with DNMT3A mutations are uniquely sensitive to the inhibition of DNA polymerase theta (Polθ encoded by POLQ gene). This effect was dependent on generation of formaldehyde by serine/one-carbon cycle metabolism. Abrogation of DNMT3A function by CRISPR/Cas9 targeting, Cre-loxP gene deletion, and heterozygous R882H mutation resulted in hypersensitivity of OTK-positive murine AML-like cells and human primary AML cells to Polθ inhibitors (Polθis) due to accumulation of toxic DSBs and activation of cGAS/STING pro-apoptotic pathway. Moreover, simultaneous loss of functional DNMT3A combined with inactivation of Polθ (by CRISPR/Cas9 targeting, insertion of neo-resistance gene, and D2230A+Y2231A polymerase inactive mutant) caused accumulation of DSBs, and reduced OTK-driven clonogenic potential and leukemogenic activity in mice. Polθ is abundantly overexpressed in OTK-positive DNMT3A-deficient cells due to enhanced POLQ mRNA stability and elevated translation of Polθ protein but not due to altered POLQ methylation and Polθ protein stability. Polθ is a key element not only in TMEJ of DSBs with limited end-resection, but also in replication fork restart and in single-strand DNA (ssDNA) gap filling. OTK-positive DNMT3A-deficient cells displayed hyperactivity of Polθ-mediated TMEJ and replication fork restart, but not ssDNA gap filling. These effects were accompanied by increased loading of Polθ on DNA damage detected by Polθ foci formation and chromatin extraction. Moreover, DNMT3A deficiency modulates chromatin architecture at DSBs to limit DNA end-resection thus favoring TMEJ over HR. Furthermore, we tested the effectiveness of Polθis combined with FDA approved drugs (quizartinib, etoposide, cytarabine, azacytidine) against FLT3(ITD)-positive DNMT3A-deficient cells (primary patient cells and cell lines) in vitro and in vivo. The combination of Polθis + quizartinib and Polθis + etoposide completely eradicated clonogenic activity of these cells while Polθis + cytarabine and Polθis + azacytidine exerted modest and weak effects, respectively, when compared to individual compound treatments. These drug combinations were only modestly toxic to normal bone marrow cells. Treatment with Polθi or etoposide reduced the percentage of GFP+ FLT3(ITD)-positive DNMT3A-deficient leukemia cells in peripheral blood of the mice by ~2-fold and prolonged survival time by ~1.5-fold. Remarkably, the combination of Polθi and etoposide eradicated leukemia cells below detectable levels in 6/12 mice with no visible toxicity. Median survival time of the mice will be recorded. Altogether, we discovered that Polθ protects OTK-positive DNMT3A-deficient myeloid malignant cells from the toxic effects of DSBs and identified Polθ as a novel therapeutic target.

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.

Monoclonal Antibodies Targeting LIGHT Block Ltbr Signalling and Eliminate Acute Myeloid Leukemia Stem Cells

Introduction Acute myeloid leukemia (AML) is a molecular heterogeneous disease that originates from the malignant transformation of a hematogenic stem- or progenitor cells resulting in a leukemia stem cell (LSC). LSCs are a main cause of resistance to therapy and relapse. Despite recent advances in targeted therapy and immunotherapy, prognosis for elderly patients not fit do undergo intensive therapy remains poor. We recently documented that targeting CD70, the ligand of the tumor necrosis factor receptor (TNFR) CD27, by the antibody-dependent cell-mediated cytotoxicity (ADCC)-enhanced monoclonal antibody (mAb) cusatuzumab eliminates LSC. We now studied the role of the related TNFR lymphotoxin b receptor (LTbR) with its ligand LIGHT in the pathogenesis of AML in mice and humans and developed a LIGHT targeting mAb to specifically eliminate LSCs. Experimental design We investigated the impact of LIGHT/LTbR signalling in AML development by using the retrovirally-induced MLL-AF9 syngeneic AML mouse model. Light-, Ltbr -deficient and control MLL-AF9 transduced LSCs were injected intravenously into non-irradiated BL/6 recipient mice. AML development was evaluated by analysing MLL-AF9-GFP-positive leukemic cells in blood, spleen, and bone marrow (BM). BM LSC were phenotypically characterized by FACS and functionally by performing colony forming unit (CFU) assays in vitro and by secondary transplantations of LSCs including limited dilution assays (LDA) in vivo. Correlative survival analysis for LIGHT and LTbR expression was performed on a publicly available gene dataset (GSE6891). LIGHT and LTBR protein expression was analysed on primary AML CD34 + leukemic stem and progenitor cells (LSPCs) and control human CD34 + hematopoietic stem cell and progenitor cells (HSCPs). LIGHT/LTbR signalling was blocked by either siRNA treatment or by treatment with novel LIGHT-targeting mAb. The effect of blocking LIGHT/LTbR-signalling was analysed in vitro by CFU assays, RNA-sequencing and xenotransplant experiments in vivo. LIGHT-targeting mAbs were generated by immunization of llamas and phage display selections followed by cloning of FABs into a human backbone (hIgG1). Clones were selected based on their blocking potency of receptor/ligand interaction using competition ELISA and FACS-based cell binding affinity assays. ADCC-enhanced anti-LIGHT mAbs were generated via POTELLIGENT® technology. Anti-LIGHT mAbs that specifically block the interaction of LIGHT/LTBR with deficiency in effector functions (Fc-death) were produced by amino acid substitutions in the CH2 domain of the parental clone. Results LTbR was expressed by HSCs and AML LSCs. In contrast, LIGHT was only expressed on murine and human LSCs but not normal HSCs. The absence of LIGHT or its receptor LTbR on LSCs resulted in significantly prolonged survival and decreased numbers of LSC in BM of AML mice. Light -/- and Ltbr -/- LSCs had a reduced CFU capacity compared to control LSCs. Similarly, inhibition of LIGHT-signalling on LSCs via LTbR-Ig significantly reduced colony formation. Moreover, secondary transplantation of Light -/-LSCs resulted in prolonged survival compared to Light-proficient LSCs. An LDA analysis revealed a 11-fold reduction of the LSC frequency in BM in the absence of LIGHT or LTBR. Similarly, inhibiting LIGHT signalling by siLIGHT RNA in human AML LSPCs resulted in reduced CFU capacity compared to control siRNA treatment. In addition, a GEO analysis revealed that low LIGHT or LTbR mRNA levels correlated with improved overall survival in AML patients. After immunizations of llamas and phage-display selection three humanized anti-LIGHT mAbs with high blocking and high binding efficiency were selected (mAb10F11 and 12C2 specific for human LIGHT and the cross-reactive murine/human 9F6 mAB). Incubation of LSCs with the blocking mAbs significantly reduced CFU capacity. HSPCs from healthy donors remained unaffected by the treatment. A gene expression analysis revealed that blocking LIGHT/LTbR-signalling in LSCs reduced stemness and promoted differentiation. In addition, ADCC-enhanced mAbs activated NK cells to eliminate LIGHT-expressing LSCs. Our findings reveal that LIGHT/LTbR-signalling is crucial for the pathogenesis of AML and especially for the maintenance and expansion of LSCs. We developed and validated novel LIGHT targeting mAbs in vitro and in vivo for further development in clinical trials.

Pegylated Long-Acting Interferon Gamma Treatment Enhances MHC Class II Expression and the Graft-Versus-Leukemia Effect in Preclinical Models of AML Relapse

Relapse after allogeneic hematopoietic stem cell transplantation (HCT)represents the most common type of treatment failure and is thought to result in part from AML cell escape from immune pressure exerted by donor immune cells (the “Graft-versus-leukemia” effect, GvL). We and others have previously reported downregulation of MHC class II (MHC-II) expression in up to half of AML cases relapsing after transplantation, but it is unclear whether a) loss of MHC-II contributes mechanistically to loss of GvL, and b) whether reversal of MHC-II downregulation can restore GvL. We tested the role of MHC-II in mediating GvL in a mouse model of minor-antigen mismatch HCT where C57BL/6 mice (B6) bearing syngeneic AML cells (H-2b) were co-transplanted with or without CD3+ T cells from C3.SW-H2b/SnJ mice (H-2b). Since murine AML cells do not generally express MHC-II at baseline, we transduced MLL-AF9-driven B6 AML cells with a fluorescently-tagged retrovirus expressing CIITA the main transcriptional regulator of MHC-II genes. MHC-II-expressing AML cells were selectively deleted when co-transplanted with donor T cells, in contrast to animals that received no T cells (Figure 1, right) or mice that received AML cells transduced with empty vector (Figure 1, left). To extend these findings to human cells, we used both MHC-II blocking antibodies and CRISPR-mediated disruption of CIITA in human AML cell lines and primary samples. Loss or blocking of MHC-II led to diminished the ability of AML cells to stimulate MHC-mismatched donor T cells in a mixed lymphocyte reaction. Loss of MHC-II expression in AML cells can be reversed in vitro by Interferon gamma (IFNG). We tested the ability of IFNG to enhance the GvL by culturing AML cell lines and primary patient samples with or without IFNG for 48 hours. IFNG-treatment enhanced the ability of AML cells from some-but not all-samples to stimulate third-party T cells. Interestingly, enhanced stimulation was observed in both CD4 and CD8 cells, suggesting that IFNG may also potentiate GvL through MHC-II-independent pathways. Since the use of IFNG in vivo may be limited by its short half-life, we tested a novel long-acting pegylated human interferon gamma (peg-IFNG, Bolder BioTechnology) in a xenograft model of AML. Immunodeficient mice were engrafted with MHC-II-low human AML cells. After engraftment, mice (n=3 each group) received six doses of IFNG, peg-IFNG, or vehicle (15mcg given s.c. M-W-F). After treatment, mice were sacrificed and MHC-II expression was quantified on human AML cells isolated from bone marrow of treated mice. IFNG treatment increased MHC-II expression modestly compared to vehicle (2.7-fold, p=0.04), while peg-IFNG much more robustly stimulated MHC-II stimulation on human AML cells in this xenograft model (12-fold, p=0.02). Finally, we tested whether peg-IFNG can enhance AML cell clearance in a xenograft model. Luciferin-labelled THP1 cells (low MHC-II expression) were injected on day 0 into immundeficient mice (n=8-9 each group). On day 7, mice started treatment with peg-IFNG or vehicle (15mcg given s.c. M-W-F for two weeks). On day 21, baseline tumor burden was measured using bioluminescence, and healthy third-party donor T cells were injected. Mice were followed with weekly CBC and bioluminescence measurments, and moribund mice were sacrificed and banked. Mice treated with peg-IFNG plus T cells had decreased tumor burden during the course of the experiment compared to untreated controls as well as mice treated with T cells plus vehicle only (Figure 2, p<0.01 by ANOVA for both comparisons). In addition, mice treated with peg-IFNG had significantly improved survival compared to control groups (median survival 79 days compared to 63 days and 67 days, p<0.05 by Log-Rank test). Together, these findings suggest that long-acting IFNG analogues may effectively enhance GvL in AML patients after HCT.

Targeting Mitochondrial Apoptotic Priming State to Personalize Therapy for Relapsed Acute Myeloid Leukemia

Despite of remarkable changes in therapeutic landscape of AML in past 5 years, relapsed AML patients still succumb to disease. In majority of patients, the common cause of death ultimately is the emergence multi-drug resistance at relapse. Using patient-derived xenograft (PDX) as our models, we investigated the basis of multi-drug resistance and methods for identifying drug sensitivity in relapsed AML. We hypothesized that there is differential activation of drug-induced mitochondrial apoptotic signaling between treatment-naïve and relapsed myeloblasts. To study mechanisms of drug resistance in AML, we developed PDX models (n=18 models) of in vivo acquired resistance to single agents (BCL-2/MCL-1 inhibitor, FLT3 inhibitor, SMAC mimetics and BRD-4 inhibitor) of distinct mechanisms, which caused board chemoresistance across all models. We tested whether multidrug resistance is associated with reduction in apoptotic signaling, using BH3 profiling, a method for measurement of mitochondrial sensitivity to apoptosis via exposure to synthetic BH3 domain peptides, on matched pre- and post-resistant PDXs. Using BH3 profiling, we found that a reduction in mitochondrial apoptotic priming consistently accompanied the acquisition of multidrug resistance irrespective of the mechanism of drug action and genetic status of PDX models. Next we created pharmacologic sensitivity landscape for 22 AML PDX models by exposing splenic myeloblasts isolated after confirmed engraftment (>70% circulating hCD45+ blasts) to drug a drug panel (n=40) for 14 hours. We first demonstrate that ex vivo drug-induced mitochondrial sensitivity singling measured via dynamic BH3 profiling (DBP) segregates PDXs according to prior treatment. By using unsupervised hierarchical clustering we found that AML PDXs developed from treatment naïve patients clustered together and showed greater apoptotic priming responses to a larger number of targeted agents than PDXs from R/R patients, which also formed discrete clusters. We replicated drug-response patterns from PDX models in primary and relapsed AML patients by analyzing ex vivo sensitivity responses to >500 drugs across relapse and diagnosis patients (Malani et al, 2021 and Tyner et al, 2018). We show that human AML patients at relapse also broadly lose sensitivity to agents in a similar pan-resistant manner as PDX models. Using unbiased whole transcriptomic analysis of naïve and resistant PDXs (n=78 samples), we found common pathways of resistance where positive enrichment was observed for cytokine-cytokine receptor interactions, xenobiotics biodegradation, metabolic pathways, Hippo signaling, and Ras signaling in resistant compared to treatment-naïve PDXs. Conversely, mechanisms relating to DNA repair and replication, such as base excision repair, mismatch repair, and homologous recombination, were negatively enriched. Moreover, pathways associated with specific drug resistance were also observed. One of the known multi-drug resistance mechanisms to chemotherapy drugs in AML is increased expression of efflux pumps from the superfamily of ATP binding cassette. We found enrichment in ABC transporter family pathway signatures in all 3 PDX models, DFAM-61786, DFAM-15354, and DFAM-61345, resistant to quizartinib, birinapant, and JQ-1 and validated higher drug-efflux activity in quizartinib and birinapant resistant model using calcein-AM efflux assay. We finally tested whether DBP could predict in vivo response of patient myeloblasts in PDX models. We tested in vivo sensitivity to 5 drugs of disparate mechanisms of action: birinapant and LCL-161 (SMAC mimetics), JQ-1 (BRD-4 inhibitor), venetoclax (BCL-2 antagonist), and quizartinib (FLT-3 inhibitor) in 4-9 different PDX models each. Using ex vivo DBP assay, we identified drugs that prime not only treatment naïve PDXs but also R/R PDXs. We identified persistent in vivo activity in a drug-specific manner, with BH3 mimetics and HDAC inhibitors showing overlapping activity across different drug-resistant models. Overall, we demonstrate that acquired resistance to targeted therapy in AML is accompanied by common mechanism of reduction in mitochondrial priming along with drug-specific resistance mechanisms. Further, we find that, even in the context of a multiply-resistant PDX model, DBP can still identify therapeutic vulnerabilities that can be efficaciously exploited in vivo.

TRIP13 Is a Fetal-Enriched Therapeutic Target in NUP98-JARID1A + Pediatric Non-Down Syndrome AMKL

Chromosomal rearrangements have been identified as the main drivers of pediatric Acute Megakaryoblastic Leukemia in absence of Down syndrome (non-DS-AMKL). The t(11;12) involving NUP98 and KDM5A/JARID1A (NJ) accounts for approximately 15% of pediatric non-DS-AMKL cases and correlates with poor prognosis. An important aspect of AMKL is the strong enrichment in pediatric patients with frequent occurrence in infants (disease within 2 years post birth). In this study, we aimed to explore the role of cellular ontogeny in NJ-driven non-DS-AMKL and highlight TRIP13 as a fetal-enriched NJ-specific vulnerability acting through P53 regulation. To investigate the mechanism of NJ transformation, we first transduced murine hematopoietic stem and progenitor cells (HSPCs) originating from fetal liver (mFL-HSPCs) and bone marrow of adult mice (mBM-HSPCs) with a lentiviral vector overexpressing the cDNA encoding human NJ. In line with other studies, NJacts as a potent oncogene in vitro, sustaining cell proliferation and arresting cell differentiation. Both mFL-HSPCs and mBM-HSPCs expressing NJ transformed and dominated the culture after 3-4 weeks with an immature immune-phenotype (Lin-, Sca1+ and c-Kit+). Notably, outgrowth of mFL-HSPCs was significantly accelerated compared to adult mBM-HSPCs, despite similar transduction rates. Furthermore, in vivo experiments revealed that NJ-expressing mFL-HSPCs displayed a more aggressive phenotype (latency of 36 days) compared to mBM-HSPCs cells expressing NJ (latency of 70 days; P Long-rank < 0.001). Thus, both our in vitro and in vivo experiments strongly indicate an impact of fetal cell background. Suspecting an impact of fetal gene programs on NJ-mediated transformation, we performed a CRISPR-Cas9 screening targeting fetal expression signatures with a library probing 880 genes found to be deregulated when comparing fetal versus adult murine and human primary HSPCs. By comparing our findings with two other fetal liver-derived leukemia models - representing DS AMKL and familial platelet disorder with predisposition to AML (FPD-AML) -, we identified TRIP13 as a high-confidence candidate gene with exclusive dependency in NJ-driven non-DS-AMKL. Comparing human AML cell lines and NJ-overexpressing human fetal liver cells (hFL-HSPCs) confirmed selective targeting of NJ-driven cells by TRIP13 loss. To explore the molecular mechanism of TRIP13 sensitivity, we performed RNAseq. Surprisingly, gene set expression analysis (GSEA) revealed one highly significantly enriched pathway, i.e. TP53 signaling. Rescue experiments of Trip13 knockout in NJ-driven non-DS-AMKL cells further supported this finding: the massive depletion upon loss of Trip13 was completely reverted by either re-expression of a human TRIP13 cDNA or by knockout of Trp53. Of note, neither activation of TP53 signaling nor cell depletion was seen in healthy hematopoietic cells from FL upon Trip13 perturbation. Finally, we aimed to leverage TRIP13 dependency therapeutically. To this end, we first explored Trip13-depletion in an in vivo setting. In a fluorescence-based competitive transplantation assay, Trip13-ablated leukemic blasts were significantly diminished in the bone marrow of recipient mice after 4 weeks, showing a mean depletion of 83%. Next, NJ-driven non-DS-AMKL mFL-HSPCs were treated with the TRIP13 inhibitor DCZ0415. Similarly to genetic TRIP13 ablation, DCZ0415-treated NJ mFL-HSPCs showed a significantly higher sensitivity to the drug compared to models of DS AMKL, FPD-AML, and healthy HSPCs with fold changes of 1.6 (p-value = 0.024), 2.3 (p-value = 0.001) and 2.0 (p-value = 0.008), respectively. To exploit our mechanistic knowledge on TRIP13 ablation-mediated P53 activation, we further combined DCZ0415 with the MDM2 inhibitor Idasanutlin. In line with our mechanistic data, a high synergy (Bliss synergy score = 9.7) of TRIP13 and MDM2 inhibition was observed. In conclusion, our study uncovers TRIP13 as a fetal-enriched vulnerability in NJ-driven non-DS-AMKL, mechanistically acting through P53, which we leveraged for a mechanism-driven treatment approach with dual TRIP13/MDM2 inhibition as a potential therapeutic strategy for the treatment of high-risk pediatric non-DS-AMKL.

Optimizing Siglec-8-Directed Immunotherapy for Eosinophilic and Mast Cell Disorders

Current treatments for diseases in which eosinophils and/or mast cells play an important pathogenic role are often insufficiently effective. One promising target for these disorders is sialic acid immunoglobulin (Ig)-like lectin 8 (Siglec-8), but there are only few monoclonal antibodies (mAbs) against Siglec-8 available, and, so far, Siglec-8-directed therapies have largely focused on unconjugated mAbs, which may lack efficacy. To raise new human mAbs as basis for novel therapeutics, we immunized transgenic (“humanized”) mice with 3T3 cells expressing human full-length Siglec-8 (Siglec-8 FL) or a chimeric mouse/human protein consisting of the V-set domain of mouse CD33 fused with the membrane-proximal C2-set domain of human Siglec-8 or the V-set domain of mouse Siglec-F fused to the membrane-proximal C2-set domain of human Siglec-8. Among 9 unique mAbs we identified and recombinantly produced, 6 (1B6, 1H4, 1B4, 1E1, 1E4, 1E2) recognized Siglec-8 via binding of the V-set domain, 1 (2A3) via binding of the membrane-distal C2-set domain, and 2 (2A7, 2F10) via binding to the membrane-proximal C2-set domain. All mAbs tested were efficiently internalized into Siglec-8+ cells (up to 75% within 4 hours), suggesting their potential to deliver cytotoxic payloads. To assess the value of Siglec-8-directed immunotherapies engaging T or NK cells, we generated tool T cell-engaging Siglec-8/CD3 bispecific antibodies (BiAbs) in the scFv-scFv format as well as Siglec-8 CAR NK cells, using binding sequences from Siglec-8 V-set mAbs. Very low concentrations (10-25 pM) of a Siglec-8/CD3 BiAb built on 1B6 elicited dose-dependent cytolytic activity against EOL-1, HL-60 as well as LUVA cells transduced with Siglec-8 FL. This BiAb also exerted Siglec-8-specific cytotoxicity against HMC 1.2 cells that were enriched for Siglec-8 expression as well as parental LUVA cells, which both display very few Siglec-8 molecules. Similarly, KHYG-1 cells transduced with a Siglec-8 V-set-directed 41BB co-stimulatory domain-containing CAR generated from scFv sequences from 1B6 or 1H4 induced significant cytotoxicity against EOL-1 and RS4;11 cells transduced with Siglec-8 FL. To examine the relationship between the efficacy of T cell and NK cell-engaging therapies and the distance from the target epitope on Siglec-8 to the cell membrane, we engineered human AML and ALL cell line pairs expressing comparable levels of either Siglec-8 FL or an artificial variant of Siglec-8 containing only the V-set domain on the extracellular portion of the protein (Siglec-8 V-set) to bring the V-set domain closer to the cell membrane, and used these cell line pairs as targets for Siglec-8 V-set-directed BiAbs and CAR-modified immune effector cells. Siglec-8 V-set/CD3 BiAbs exerted greater cytotoxicity against AML and ALL cells expressing Siglec-8 V-set than cells expressing Siglec-8 FL. Likewise, Siglec-8 V-set-directed CAR NK cells exerted greater cytotoxicity against AML and ALL cells expressing Siglec-8 V-set than cells expressing Siglec-8 FL, demonstrating that altering the position of the Siglec-8 mAb binding epitope changes the effector functions of Siglec-8 mAb-derived therapies and suggesting membrane-proximal targeting via one of the C2-set domains could enhance Siglec-8-targeted therapies that engage T or NK cells as immune effector cells. To test whether mAbs recognizing one of the C2-set domains could indeed serve as basis for potent therapeutics, we generated a Siglec-8 C2-set/CD3 BiAb in the scFv-scFv format using binding sequences from 2A3 and found it effectively killed EOL-1, ML-1, RS4;11, and LUVA cells transduced with human Siglec-8 FL at very low concentrations. This BiAb also exerted Siglec-8-specific cytotoxicity against Siglec-8-enriched HMC 1.2 cells and parental LUVA cells, which both display Siglec-8 at very low copy number. Likewise, KHYG-1 cells transduced with a Siglec-8 C2-set CAR generated from 2A3 or 2F10 induced significant cytotoxicity against EOL-1 and RS4;11 cells transduced with Siglec-8 FL as well as against Siglec-8-enriched HMC 1.2 cells, albeit to a very modest extent. Together, our data demonstrate Siglec-8-directed immunotherapies can be highly potent, even against target cells expressing very limited copy numbers of Siglec-8 molecules, supporting their further development for use in patients with difficult-to-treat eosinophilic and mast cell disorders.

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.

Insight into the mechanism of AML del(9q) progression: hnRNP K targets the myeloid master regulators CEBPA (C/EBPα) and SPI1 (PU.1)

Deletions on the long arm of chromosome 9 (del(9q)) are recurrent abnormalities in about 2 % of acute myeloid leukemia cases, which usually involve HNRNPK and are frequently associated with other known aberrations. Based on an Hnrnpk haploinsufficient mouse model, a recent study demonstrated a function of hnRNP K in pathogenesis of myeloid malignancies via the regulation of cellular proliferation and myeloid differentiation programs. Here, we provide evidence that reduced hnRNP K expression results in the dysregulated expression of C/EBPα and additional transcription factors. CyTOF analysis revealed monocytic skewing with increased levels of mature myeloid cells. To explore the role of hnRNP K during normal and pathological myeloid differentiation in humans, we characterized hnRNP K-interacting RNAs in human AML cell lines. Notably, RNA-sequencing revealed several mRNAs encoding key transcription factors involved in the regulation of myeloid differentiation as targets of hnRNP K. We showed that specific sequence motifs confer the interaction of SPI1 and CEBPA 5′ and 3′UTRs with hnRNP K. The siRNA mediated reduction of hnRNP K in human AML cells resulted in an increase of PU.1 and C/EBPα that is most pronounced for the p30 isoform. The combinatorial treatment with the inducer of myeloid differentiation valproic acid resulted in increased C/EBPα expression and myeloid differentiation. Together, our results indicate that hnRNP K post-transcriptionally regulates the expression of myeloid master transcription factors. These novel findings can inaugurate novel options for targeted treatment of AML del(9q) by modulation of hnRNP K function.

Regulation of Metabolic Homeostasis By TRAF6 Contributes to the Leukemia Progression

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that mediates innate immune signaling that converges on key mediators of Toll-like receptor and interleukin-1 signaling. Dysregulated TRAF6 expression and innate immune pathway activation is implicated in the pathogenesis of various myeloid malignancies. For instance, increased TRAF6-mediated signaling in hematopoietic stem and progenitor cells (HSPCs) results in myelodysplastic syndrome-like cellular features, such as the functional impairment of HSPCs. On the other hand, our previous study indicated that loss of TRAF6 induces the leukemic transformation of pre-leukemic cells ( Muto et al. Cell Stem Cell). These paradoxical observations suggests that TRAF6 acts as both an oncogene and tumor suppressor gene in hematopoietic cells in a disease type- and context-dependent manner. However, the direct consequence of TRAF6 loss in AML cells, rather than in pre-leukemic cells, has not yet been explored. Therefore, the role of TRAF6 in the pathogenesis of AML remains obscure. In this study, we found elevated level of TRAF6 expression in AML cells, and determined the effect of TRAF6 loss on the cellular and molecular features of AML cells. We proposed a novel role of TRAF6 as a regulator of metabolic homeostasis, which is important for the progression of AML. To determine the requirement of TRAF6 in AML, we first evaluated cell the effect of TRAF6 loss in AML cell lines on proliferative capacities. Multiple types of human AML cell lines showed significant growth impairment upon TRAF6-knockdown. Furthermore, transplantation of TRAF6-deficient HSPCs expressing the leukemic oncogene MLL-AF9 to the recipient mice resulted in suppression of overt leukemia. These findings confirm that loss of TRAF6 suppresses leukemic cell function in vitro and in vivo. To assess the molecular mechanism of inhibitory effect of TRAF6 loss in AML, we next performed gene expression analysis using human AML database and TRAF6-knockdown AML cell lines. Interestingly, we found that reduced levels of TRAF6 in AML is associated with changes in the expression of genes involved in mitochondria-related processes. Our extensive in vitro validation experiments including extracellular flux assay and metabolome analysis confirmed that TRAF6 loss in AML cells induces dynamic changes in metabolites and the impairment of mitochondrial function. To identify the mechanism of metabolic alterations by TRAF6 loss in leukemia, we compared the upregulated genes in TRAF6-knockdown AML cells with the 130 previously identified essential genes for AML cell survival in vitro and in vivo ( Yamauchi et al. Cancer Cell). We focused on O-GlcNAc transferase ( OGT) as a gene associated in mitochondrial function and AML cell survival. Importantly, TRAF6 gene expression in human AML patient samples was positively correlated with the expression of OGT. To confirm the importance of OGT in the regulation of mitochondrial function mediated by TRAF6 in leukemia, we utilized the inhibitor of O-GlcNAcase (OGA), a remover of O-GlcNAc from its substrates. The treatment with MK8719 (OGA inhibitor) mildly, but significantly, restored the number of TRAF6 knockdown AML cells, which was correlated with the changes in mitochondrial function, indicating that O-GlcNAc modification regulated by TRAF6 is important for AML progression. In summary, We provide evidence for the oncogenic function of TRAF6 in leukemia, and shed light on the novel TRAF6/OGT/O-GlcNAc axis that regulates the metabolic reprogramming required for leukemogenesis.