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Targeting AML by chimeric antigen receptor T-cells (CAR-T) is challenging due to the promiscuous expression of AML-associated antigens in healthy hematopoiesis and high degree of inter- and intratumoral heterogeneity. Here, we present single-cell expression data of AML-associated antigens in 30 primary pediatric AML samples. We identified CD33, CD38, CD371, IL1RAP and CD123 as the most frequently expressed. Notably, high variability was observed not only across the different patient samples but also among leukemic cells of the same patient suggesting the necessity of multiplexed targeting approaches. To address this need, we utilized our modular Adapter CAR (AdCAR) platform, enabling precise qualitative and quantitative control over CAR-T-cell function. We show highly efficient and target-specific activity for newly generated adapter molecules (AMs) against CD33, CD38, CD123, CD135 and CD371, both in vitro and in vivo. We reveal that inherent intratumoral heterogeneity in antigen expression translates into antigen escape and therapy failure to monotargeted CAR-T therapy. Further, we demonstrate in PDX models that rational combinatorial targeting by AdCAR-T-cells can cure heterogenic disease. In conclusion, we elucidate the clinical relevance of heterogeneity in antigen expression in pediatric AML and present a novel concept for precision immunotherapy by combinatorial targeting utilizing the AdCAR platform., (© 2024. The Author(s).)

Abstract: Aberrant expression of stem cell-associated genes is a common feature in acute myeloid leukemia (AML) and is linked to leukemic self-renewal and therapy resistance. Using AF10-rearranged leukemia as a prototypical example of the recurrently activated "stemness" network in AML, we screened for chromatin regulators that sustain its expression. We deployed a CRISPR-Cas9 screen with a bespoke domain-focused library and identified several novel chromatin-modifying complexes as regulators of the TALE domain transcription factor MEIS1, a key leukemia stem cell (LSC)-associated gene. CRISPR droplet sequencing revealed that many of these MEIS1 regulators coordinately controlled the transcription of several AML oncogenes. In particular, we identified a novel role for the Tudor-domain-containing chromatin reader protein SGF29 in the transcription of AML oncogenes. Furthermore, SGF29 deletion impaired leukemogenesis in models representative of multiple AML subtypes in multiple AML subtype models. Our studies reveal a novel role for SGF29 as a nononcogenic dependency in AML and identify the SGF29 Tudor domain as an attractive target for drug discovery., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Chimeric antigen receptor T cells (CAR-T cells) have emerged as a powerful treatment option for individuals with B cell malignancies but have yet to achieve success in treating acute myeloid leukemia (AML) due to a lack of safe targets. Here we leveraged an atlas of publicly available RNA-sequencing data of over 500,000 single cells from 15 individuals with AML and tissue from 9 healthy individuals for prediction of target antigens that are expressed on malignant cells but lacking on healthy cells, including T cells. Aided by this high-resolution, single-cell expression approach, we computationally identify colony-stimulating factor 1 receptor and cluster of differentiation 86 as targets for CAR-T cell therapy in AML. Functional validation of these established CAR-T cells shows robust in vitro and in vivo efficacy in cell line- and human-derived AML models with minimal off-target toxicity toward relevant healthy human tissues. This provides a strong rationale for further clinical development., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Fms-like tyrosine kinase 3 (FLT3) is often overexpressed or constitutively activated by internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations in acute myeloid leukemia (AML). Despite the use of receptor tyrosine kinase inhibitors (TKI) in FLT3-ITD-positive AML, the prognosis of patients is still poor, and further improvement of therapy is required. Targeting FLT3 independent of mutations by antibody-drug conjugates (ADCs) is a promising strategy for AML therapy. Here, we report the development and preclinical characterization of a novel FLT3-targeting ADC, 20D9-ADC, which was generated by applying the innovative P5 conjugation technology. In vitro, 20D9-ADC mediated potent cytotoxicity to Ba/F3 cells expressing transgenic FLT3 or FLT3-ITD, to AML cell lines, and to FLT3-ITD-positive patient-derived xenograft AML cells. In vivo, 20D9-ADC treatment led to a significant tumor reduction and even durable complete remission in AML xenograft models. Furthermore, 20D9-ADC demonstrated no severe hematotoxicity in in vitro colony formation assays using concentrations that were cytotoxic in AML cell line treatment. The combination of 20D9-ADC with the TKI midostaurin showed strong synergy in vitro and in vivo, leading to reduction of aggressive AML cells below the detection limit. Our data indicate that targeting FLT3 with an advanced new-generation ADC is a promising and potent antileukemic strategy, especially when combined with FLT3-TKI in FLT3-ITD-positive AML., (© 2023 by The American Society of Hematology.)

The development and regulation of malignant self-renewal remain unresolved issues. Here, we provide biochemical, genetic, and functional evidence that dynamics in ribosomal RNA (rRNA) 2'-O-methylation regulate leukemia stem cell (LSC) activity in vivo. A comprehensive analysis of the rRNA 2'-O-methylation landscape of 94 patients with acute myeloid leukemia (AML) revealed dynamic 2'-O-methylation specifically at exterior sites of ribosomes. The rRNA 2'-O-methylation pattern is closely associated with AML development stage and LSC gene expression signature. Forced expression of the 2'-O-methyltransferase fibrillarin (FBL) induced an AML stem cell phenotype and enabled engraftment of non-LSC leukemia cells in NSG mice. Enhanced 2'-O-methylation redirected the ribosome translation program toward amino acid transporter mRNAs enriched in optimal codons and subsequently increased intracellular amino acid levels. Methylation at the single site 18S-guanosine 1447 was instrumental for LSC activity. Collectively, our work demonstrates that dynamic 2'-O-methylation at specific sites on rRNAs shifts translational preferences and controls AML LSC self-renewal., Significance: We establish the complete rRNA 2'-O-methylation landscape in human AML. Plasticity of rRNA 2'-O-methylation shifts protein translation toward an LSC phenotype. This dynamic process constitutes a novel concept of how cancers reprogram cell fate and function. This article is highlighted in the In This Issue feature, p. 247., (©2022 The Authors; Published by the American Association for Cancer Research.)

BCL-2 inhibition has been shown to be effective in acute myeloid leukemia (AML) in combination with hypomethylating agents or low-dose cytarabine. However, resistance and relapse represent major clinical challenges. Therefore, there is an unmet need to overcome resistance to current venetoclax-based strategies. We performed high-throughput drug screening to identify effective combination partners for venetoclax in AML. Overall, 64 antileukemic drugs were screened in 31 primary high-risk AML samples with or without venetoclax. Gilteritinib exhibited the highest synergy with venetoclax in FLT3 wild-type AML. The combination of gilteritinib and venetoclax increased apoptosis, reduced viability, and was active in venetoclax-azacitidine-resistant cell lines and primary patient samples. Proteomics revealed increased FLT3 wild-type signaling in specimens with low in vitro response to the currently used venetoclax-azacitidine combination. Mechanistically, venetoclax with gilteritinib decreased phosphorylation of ERK and GSK3B via combined AXL and FLT3 inhibition with subsequent suppression of the antiapoptotic protein MCL-1. MCL-1 downregulation was associated with increased MCL-1 phosphorylation of serine 159, decreased phosphorylation of threonine 161, and proteasomal degradation. Gilteritinib and venetoclax were active in an FLT3 wild-type AML patient-derived xenograft model with TP53 mutation and reduced leukemic burden in 4 patients with FLT3 wild-type AML receiving venetoclax-gilteritinib off label after developing refractory disease under venetoclax-azacitidine. In summary, our results suggest that combined inhibition of FLT3/AXL potentiates venetoclax response in FLT3 wild-type AML by inducing MCL-1 degradation. Therefore, the venetoclax-gilteritinib combination merits testing as a potentially active regimen in patients with high-risk FLT3 wild-type AML., (© 2022 by The American Society of Hematology.)

Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25 % of acute myeloid leukemia (AML) patients, drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and occurs in the absence of identifiable genetic causes. This suggests that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. Here, we systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD + AML using recently developed functional translatome proteomics (measuring changes in the nascent proteome) with phosphoproteomics. This pinpointed AKT-mTORC1-ULK1-dependent autophagy as a dominant resistance mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD + cells in vitro and in primary human AML cells ex vivo. Pharmacological or genetic inhibition of autophagy increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In mice xenografted with FLT3-ITD + AML cells, co-treatment with oral FLT3 and autophagy inhibitors synergistically impaired leukemia progression and extended overall survival. Our findings identify a molecular mechanism responsible for primary FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction., (© 2022. The Author(s).)

Antibody-based immunotherapy is a promising strategy for targeting chemoresistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated by using CrossMAb and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms tumor protein (WT1) in the context of HLA-A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary acute myeloid leukemia (AML) cells was mediated in ex vivo long-term cocultures by using allogeneic (mean ± standard error of the mean [SEM] specific lysis, 67 ± 6% after 13-14 days; n = 18) or autologous, patient-derived T cells (mean ± SEM specific lysis, 54 ± 12% after 11-14 days; n = 8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean ± SEM specific lysis on days 3-4, 45.4 ± 9.0% vs 70.8 ± 8.3%; P = .015; n = 9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors exhibited a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo, and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase 1 trial in patients with relapsed/refractory AML (#NCT04580121)., (© 2021 by The American Society of Hematology.)

Background: Acute myeloid leukaemia (AML) stem cells (LSCs) cause disease relapse. The CD47 "don't eat me signal" is upregulated on LSCs and contributes to immune evasion by inhibiting phagocytosis through interacting with myeloid-specific signal regulatory protein alpha (SIRPα). Activation of macrophages by blocking CD47 has been successful, but the ubiquitous expression of CD47 on healthy cells poses potential limitations for such therapies. In contrast, CD123 is a well-known LSC-specific surface marker utilized as a therapeutic target. Here, we report the development of SIRPα-αCD123 fusion antibodies that localize the disruption of CD47/SIRPα signalling to AML while specifically enhancing LSC clearance., Methods: SIRPα-αCD123 antibodies were generated by fusing the extracellular domain of SIRPα to an αCD123 antibody. The binding properties of the antibodies were analysed by flow cytometry and surface plasmon resonance. The functional characteristics of the fusion antibodies were determined by antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity assays using primary AML patient cells. Finally, an in vivo engraftment assay was utilized to assess LSC targeting., Results: SIRPα-αCD123 fusion antibodies exhibited increased binding and preferential targeting of CD123 + CD47 + AML cells even in the presence of CD47 + healthy cells. Furthermore, SIRPα-αCD123 fusion antibodies confined disruption of the CD47-SIRPα axis locally to AML cells. In vitro experiments demonstrated that SIRPα-αCD123 antibodies greatly enhanced AML cell phagocytosis mediated by allogeneic and autologous macrophages. Moreover, SIRPα-αCD123 fusion antibodies efficiently targeted LSCs with in vivo engraftment potential., Conclusions: SIRPα-αCD123 antibodies combine local CD47 blockade with specific LSC targeting in a single molecule, minimize the risk of targeting healthy cells and efficiently eliminate AML LSCs. These results validate SIRPα-αCD123 antibodies as promising therapeutic interventions for AML., (© 2021. The Author(s).)

High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ER T2 -loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients' leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future., (© 2021. The Author(s).)

Acute myeloid leukemia (AML) is a rapidly progressing cancer, for which chemotherapy remains standard treatment and additional therapeutic targets are requisite. Here, we show that AML cells secrete the stem cell growth factor R-spondin 2 (RSPO2) to promote their self-renewal and prevent cell differentiation. Although RSPO2 is a well-known WNT agonist, we reveal that it maintains AML self-renewal WNT independently, by inhibiting BMP receptor signaling. Autocrine RSPO2 signaling is also required to prevent differentiation and to promote self-renewal in normal hematopoietic stem cells as well as primary AML cells. Comprehensive datamining reveals that RSPO2 expression is elevated in patients with AML of poor prognosis. Consistently, inhibiting RSPO2 prolongs survival in AML mouse xenograft models. Our study indicates that in AML, RSPO2 acts as an autocrine BMP antagonist to promote cancer cell renewal and may serve as a marker for poor prognosis., Competing Interests: Declaration of interests C.N., R.S., and H.L. are listed in a pending patent based on this study., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

N 6 -methyladenosine (m 6 A) is an abundant internal RNA modification 1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex 3,4 . The m 6 A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown 5-7 . Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m 6 A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.

Chemotherapy resistance is the main impediment in the treatment of acute myeloid leukaemia (AML). Despite rapid advances, the various mechanisms inducing resistance development remain to be defined in detail. Here we report that loss-of-function mutations (LOF) in the histone methyltransferase EZH2 have the potential to confer resistance against the chemotherapeutic agent cytarabine. We identify seven distinct EZH2 mutations leading to loss of H3K27 trimethylation via multiple mechanisms. Analysis of matched diagnosis and relapse samples reveal a heterogenous regulation of EZH2 and a loss of EZH2 in 50% of patients. We confirm that loss of EZH2 induces resistance against cytarabine in the cell lines HEK293T and K562 as well as in a patient-derived xenograft model. Proteomics and transcriptomics analysis reveal that resistance is conferred by upregulation of multiple direct and indirect EZH2 target genes that are involved in apoptosis evasion, augmentation of proliferation and alteration of transmembrane transporter function. Our data indicate that loss of EZH2 results in upregulation of its target genes, providing the cell with a selective growth advantage, which mediates chemotherapy resistance.

Acute myeloid leukemia (AML) is an aggressive disease for which only few targeted therapies are available. Using high-throughput RNA interference (RNAi) screening in AML cell lines, we identified LIM kinase 1 (LIMK1) as a potential novel target for AML treatment. High LIMK1 expression was significantly correlated with shorter survival of AML patients and coincided with FLT3 mutations, KMT2A rearrangements, and elevated HOX gene expression. RNAi- and CRISPR-Cas9-mediated suppression as well as pharmacologic inhibition of LIMK1 and its close homolog LIMK2 reduced colony formation and decreased proliferation due to slowed cell-cycle progression of KMT2A-rearranged AML cell lines and patient-derived xenograft (PDX) samples. This was accompanied by morphologic changes indicative of myeloid differentiation. Transcriptome analysis showed upregulation of several tumor suppressor genes as well as downregulation of HOXA9 targets and mitosis-associated genes in response to LIMK1 suppression, providing a potential mechanistic basis for the anti-leukemic phenotype. Finally, we observed a reciprocal regulation between LIM kinases (LIMK) and CDK6, a kinase known to be involved in the differentiation block of KMT2A-rearranged AML, and addition of the CDK6 inhibitor palbociclib further enhanced the anti-proliferative effect of LIMK inhibition. Together, these data suggest that LIMK are promising targets for AML therapy.

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic immune receptor sensing viral RNA. It triggers the release of type I interferons (IFN) and proinflammatory cytokines inducing an adaptive cellular immune response. We investigated the therapeutic potential of systemic RIG-I activation by short 5'-triphosphate-modified RNA (ppp-RNA) for the treatment of acute myeloid leukemia (AML) in the syngeneic murine C1498 AML tumor model. ppp-RNA treatment significantly reduced tumor burden, delayed disease onset and led to complete remission including immunological memory formation in a substantial proportion of animals. Therapy-induced tumor rejection was dependent on CD4 + and CD8 + T cells, but not on NK or B cells, and relied on intact IFN and mitochondrial antiviral signaling protein (MAVS) signaling in the host. Interestingly, ppp-RNA treatment induced programmed death ligand 1 (PD-L1) expression on AML cells and established therapeutic sensitivity to anti-PD-1 checkpoint blockade in vivo. In immune-reconstituted humanized mice, ppp-RNA treatment reduced the number of patient-derived xenografted (PDX) AML cells in blood and bone marrow while concomitantly enhancing CD3 + T cell counts in the respective tissues. Due to its ability to establish a state of full remission and immunological memory, our findings show that ppp-RNA treatment is a promising strategy for the immunotherapy of AML.

ZBTB7A is frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. However, the oncogenic collaboration between mutated ZBTB7A and the RUNX1-RUNX1T1 fusion gene in AML t(8;21) remains unclear. Here, we investigate the role of ZBTB7A and its mutations in the context of normal and malignant hematopoiesis. We demonstrate that clinically relevant ZBTB7A mutations in AML t(8;21) lead to loss of function and result in perturbed myeloid differentiation with block of the granulocytic lineage in favor of monocytic commitment. In addition, loss of ZBTB7A increases glycolysis and hence sensitizes leukemic blasts to metabolic inhibition with 2-deoxy-D-glucose. We observed that ectopic expression of wild-type ZBTB7A prevents RUNX1-RUNX1T1-mediated clonal expansion of human CD34+ cells, whereas the outgrowth of progenitors is enabled by ZBTB7A mutation. Finally, ZBTB7A expression in t(8;21) cells lead to a cell cycle arrest that could be mimicked by inhibition of glycolysis. Our findings suggest that loss of ZBTB7A may facilitate the onset of AML t(8;21), and that RUNX1-RUNX1T1-rearranged leukemia might be treated with glycolytic inhibitors.

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy leading to initial treatment responses, relapse caused by intrinsic or acquired drug resistance represents a major challenge. Here, we report that histone 3 lysine 27 demethylase KDM6A (UTX) is targeted by inactivating mutations and mutation-independent regulation in relapsed AML. Analyses of matched diagnosis and relapse specimens from individuals with KDM6A mutations showed an outgrowth of the KDM6A mutated tumor population at relapse. KDM6A expression is heterogeneously regulated and relapse-specific loss of KDM6A was observed in 45.7% of CN-AML patients. KDM6A-null myeloid leukemia cells were more resistant to treatment with the chemotherapeutic agents cytarabine (AraC) and daunorubicin. Inducible re-expression of KDM6A in KDM6A-null cell lines suppressed proliferation and sensitized cells again to AraC treatment. RNA expression analysis and functional studies revealed that resistance to AraC was conferred by downregulation of the nucleoside membrane transporter ENT1 (SLC29A1) by reduced H3K27 acetylation at the ENT1 locus. Our results show that loss of KDM6A provides cells with a selective advantage during chemotherapy, which ultimately leads to the observed outgrowth of clones with KDM6A mutations or reduced KDM6A expression at relapse.

FLT3, DNMT3A , and NPM1 are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific GPR56 high CD34 low immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor ( HLF ). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from DNMT3A/NPM1 double-mutated subclones. Moreover, in DNMT3A R882-mutated patients, CpG hypomethylation at the HLF transcription start site correlated with high HLF mRNA expression, which was itself associated with poor survival. Loss of HLF via CRISPR/Cas9 significantly reduced the CD34 + GPR56 + LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. HLF knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when reintroduced in culture. RNA sequencing of primary human triple-mutated AML cells after shRNA-mediated HLF knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 ( HES1 ) and the cyclin-dependent kinase inhibitor CDKN1C/p57 as novel targets of HLF, potentially mediating these effects. Overall, our data establish HLF as a novel LSC regulator in this genetically defined high-risk AML subgroup., (© 2019 by The American Society of Hematology.)

Abnormal metabolism is a fundamental hallmark of cancer and represents a therapeutic opportunity, yet its regulation by oncogenes remains poorly understood. Here, we uncover that JMJD1C, a jumonji C (JmjC)-containing H3K9 demethylase, is a critical regulator of aberrant metabolic processes in homeobox A9 (HOXA9)-dependent acute myeloid leukemia (AML). JMJD1C overexpression increases in vivo cell proliferation and tumorigenicity through demethylase-independent upregulation of a glycolytic and oxidative program, which sustains leukemic cell bioenergetics and contributes to an aggressive AML phenotype in vivo. Targeting JMJD1C-mediated metabolism via pharmacologic inhibition of glycolysis and oxidative phosphorylation led to ATP depletion, induced necrosis/apoptosis and decreased tumor growth in vivo in leukemias co-expressing JMJD1C and HOXA9. The anti-metabolic therapy effectively diminished AML stem/progenitor cells and reduced tumor burden in a primary AML patient-derived xenograft. Our data establish a direct link between drug responses and endogenous expression of JMJD1C and HOXA9 in human AML cell line- and patient-derived xenografts. These findings demonstrate a previously unappreciated role for JMJD1C in counteracting adverse metabolic changes and retaining the metabolic integrity during tumorigenesis, which can be exploited therapeutically.

We recently identified the splicing kinase gene SRPK1 as a genetic vulnerability of acute myeloid leukemia (AML). Here, we show that genetic or pharmacological inhibition of SRPK1 leads to cell cycle arrest, leukemic cell differentiation and prolonged survival of mice transplanted with MLL-rearranged AML. RNA-seq analysis demonstrates that SRPK1 inhibition leads to altered isoform levels of many genes including several with established roles in leukemogenesis such as MYB, BRD4 and MED24. We focus on BRD4 as its main isoforms have distinct molecular properties and find that SRPK1 inhibition produces a significant switch from the short to the long isoform at the mRNA and protein levels. This was associated with BRD4 eviction from genomic loci involved in leukemogenesis including BCL2 and MYC. We go on to show that this switch mediates at least part of the anti-leukemic effects of SRPK1 inhibition. Our findings reveal that SRPK1 represents a plausible new therapeutic target against AML.

The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regard to oncogenic alterations that do not only serve as prognostic markers but also as therapeutic targets in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Because of sequence-dependent protein conformational changes FLT3-ITD tends to autophosphorylate and displays a constitutive intracellular localization. Here, we analyzed the effect of tyrosine kinase inhibitors (TKIs) on the localization of the FLT3 receptor and its mutants. TKI treatment increased the surface expression through upregulation of FLT3 and glycosylation of FLT3-ITD and FLT3-D835Y mutants. In T cell-mediated cytotoxicity (TCMC) assays, using a bispecific FLT3 × CD3 antibody construct, the combination with TKI treatment increased TCMC in the FLT3-ITD-positive AML cell lines MOLM-13 and MV4-11, patient-derived xenograft cells and primary patient samples. Our findings provide the basis for rational combination of TKI and FLT3-directed immunotherapy with potential benefit for FLT3-ITD-positive AML patients.

C-X-C chemokine receptor 4 (CXCR4) is a transmembrane receptor with pivotal roles in cell homing and hematopoiesis. CXCR4 is also involved in survival, proliferation and dissemination of cancer, including acute lymphoblastic and myeloid leukemia (ALL, AML). Relapsed/refractory ALL and AML are frequently resistant to conventional therapy and novel highly active strategies are urgently needed to overcome resistance. Methods: We used patient-derived (PDX) and cell line-based xenograft mouse models of ALL and AML to evaluate the efficacy and toxicity of a CXCR4-targeted endoradiotherapy (ERT) theranostic approach. Results: The positron emission tomography (PET) tracer 68 Ga-Pentixafor enabled visualization of CXCR4 positive leukemic burden. In xenografts, CXCR4-directed ERT with 177 Lu-Pentixather distributed to leukemia harboring organs and resulted in efficient reduction of leukemia. Despite a substantial in vivo cross-fire effect to the leukemia microenvironment, mesenchymal stem cells (MSCs) subjected to ERT were viable and capable of supporting the growth and differentiation of non-targeted normal hematopoietic cells ex vivo . Finally, three patients with refractory AML after first allogeneic hematopoietic stem cell transplantation (alloSCT) underwent CXCR4-directed ERT resulting in leukemia clearance, second alloSCT, and successful hematopoietic engraftment. Conclusion: Targeting CXCR4 with ERT is feasible and provides a highly efficient means to reduce refractory acute leukemia for subsequent cellular therapies. Prospective clinical trials testing the incorporation of CXCR4 targeting into conditioning regimens for alloSCT are highly warranted., Competing Interests: Competing Interests: H.J.W. is shareholder of Scintomics (Germany). All other authors have no relevant conflicts of interest to declare.

In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction of histone H3K27 trimethylation as a novel pathway of acquired resistance to tyrosine kinase inhibitors (TKIs) and cytotoxic drugs in AML. Low EZH2 protein levels correlated with poor prognosis in AML patients. Suppression of EZH2 protein expression induced chemoresistance of AML cell lines and primary cells in vitro and in vivo. Low EZH2 levels resulted in derepression of HOX genes, and knockdown of HOXB7 and HOXA9 in the resistant cells was sufficient to improve sensitivity to TKIs and cytotoxic drugs. The endogenous loss of EZH2 expression in resistant cells and primary blasts from a subset of relapsed AML patients resulted from enhanced CDK1-dependent phosphorylation of EZH2 at Thr487. This interaction was stabilized by heat shock protein 90 (HSP90) and followed by proteasomal degradation of EZH2 in drug-resistant cells. Accordingly, inhibitors of HSP90, CDK1 and the proteasome prevented EZH2 degradation, decreased HOX gene expression and restored drug sensitivity. Finally, patients with reduced EZH2 levels at progression to standard therapy responded to the combination of bortezomib and cytarabine, concomitant with the re-establishment of EZH2 expression and blast clearance. These data suggest restoration of EZH2 protein as a viable approach to overcome treatment resistance in this AML patient population.

Bispecific T-cell engagers (BiTEs) are very effective in recruiting and activating T cells. We tested the cytotoxicity of the CD33/CD3 BiTE antibody construct AMG 330 on primary acute myeloid leukemia (AML) cells ex vivo and characterized parameters contributing to antileukemic cytolytic activity. The E:T ratio and the CD33 expression level significantly influenced lysis kinetics in long-term cultures of primary AML cells (n=38). AMG 330 induced T-cell-mediated proinflammatory conditions, favoring the upregulation of immune checkpoints on target and effector cells. Although not constitutively expressed at the time of primary diagnosis (n=123), PD-L1 was strongly upregulated on primary AML cells upon AMG 330 addition to ex vivo cultures (n=27, P<0.0001). This phenomenon was cytokine-driven as the sole addition of interferon (IFN)-γ and tumor necrosis factor-α also induced expression. Through blockade of the PD-1/PD-L1 interaction, AMG 330-mediated lysis (n=9, P=0.03), T-cell proliferation (n=9, P=0.01) and IFN-γ secretion (n=8, P=0.008) were significantly enhanced. The combinatorial approach was most beneficial in settings of protracted AML cell lysis. Taken together, we have characterized a critical resistance mechanism employed by primary AML cells under AMG 330-mediated proinflammatory conditions. Our results support the evaluation of checkpoint molecules in upcoming clinical trials with AMG 330 to enhance BiTE antibody construct-mediated cytotoxicity.

In acute myeloid leukemia (AML), several signaling pathways such as the phosphatidylinositol-3-kinase/AKT and the mammalian target of rapamycin (PI3K/AKT/mTOR) pathway are deregulated and constitutively activated as a consequence of genetic and cytogenetic abnormalities. We tested the effectiveness of PI3K/AKT/mTOR-targeting therapies and tried to identify alterations that associate with treatment sensitivity. By analyzing primary samples and cell lines, we observed a wide range of cytotoxic activity for inhibition of AKT (MK-2206), mTORC1 (rapamycin) and PI3K/mTORC1/2 (BEZ-235) with a high sensitivity of cells carrying an MLL rearrangement. In vivo PI3K/mTOR inhibition delayed tumor progression, reduced tumor load and prolonged survival in an MLL-AF9(+)/FLT3-ITD(+) xenograft mouse model. By performing targeted amplicon sequencing in 38 MLL-AF9(+) and 125 cytogenetically normal AML patient samples, we found a high additional mutation rate for genes involved in growth factor signaling in 79% of all MLL-AF9(+) samples, which could lead to a possible benefit of this cohort. PI3K/mTOR inhibition for 24 h led to the cross-activation of the ERK pathway. Further in vitro studies combining PI3K/mTOR and ERK pathway inhibition revealed highly synergistic effects in apoptosis assays. Our data implicate a possible therapeutic benefit of PI3K/mTOR inhibition in the MLL-mutated subgroup. Inhibiting rescue pathways could improve the therapeutic efficacy of PI3K-targeted therapies in AML.

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups.

Application of anthracyclines and Vinca alkaloids on the same day represents a hallmark of polychemotherapy protocols for hematopoietic malignancies. Here we show, for the first time, that both drugs might act most efficiently if they are applied on different days. Proof-of-concept studies in 18 cell lines revealed that anthracyclines inhibited cell death by Vinca alkaloids in 83% of cell lines. Importantly, in a preclinical mouse model, doxorubicin reduced the anti-tumor effect of vincristine. Both drugs acted in a sequence-dependent manner and the strongest anti-tumor effect was obtained if both drugs were applied on different days. Most notably for clinical relevance, in 34% of 35 fresh primary childhood leukemia cells tested in vitro, doxorubicin reduced the anti-tumor effect of vincristine. As underlying mechanism, doxorubicin activated p53, p53 induced cell-cycle arrest, and cell-cycle arrest disabled inactivation of antiapoptotic Bcl-2 family members by vincristine; therefore, vincristine was unable to activate downstream apoptosis signaling. As molecular proof, antagonism was rescued by knockdown of p53, whereas knockdown of cyclin A inhibited vincristine-induced apoptosis. Our data suggest evaluating anthracyclines and Vinca alkaloids on different days in future trials. Selecting drug combinations based on mechanistic understanding represents a novel conceptional strategy for potent polychemotherapy protocols.