Leukemic Progenitor Research Articles

RNA Editing By APOBEC3A Confers Dynamic Mutational Load in Monocytic Leukemias

Background: Although genomic mutations play an important role in the initiation or progression of myeloid leukemias, the role of mutations at the transcript level is less well understood. Myeloid neoplasms have recurrent somatic mutations in genes for pre-mRNA splicing, cohesin complex, histone modification, and DNA methylation. However, the number of somatic mutations identified in myeloid neoplasms (average 9-13) is usually lower than that of solid tumors. Recent analyses of baseline transcriptome sequences in various cancers have revealed abundant adenosine to inosine (A>I) RNA editing events, mainly at intronic and untranslated regions of genes. The role of condition-specific RNA editing in cancer, especially of the cytosine to uracil (C>U) type, however, remains essentially unknown. We recently discovered antiviral restriction enzyme APOBEC3A (A3A) cytidine deaminase as a novel cellular site-specific C>U RNA editing enzyme in monocytes. RNA editing by A3A is dynamically induced by interferons and/or severe hypoxia in monocytes, resulting in stop codon gains or missense changes in transcripts of scores of genes. The effect of hypoxia in inducing RNA editing by A3A can be mimicked by inhibition of mitochondrial respiration. Whether A3A-mediated RNA editing occurs in monocytic leukemias, including acute monocytic leukemia (AMoL) and chronic myelomonocytic leukemia (CMML), is unknown. We hypothesize that dynamic C>U RNA editing by A3Acontributes to mutational load of leukemic cells under stressful conditions of hypoxia and interferon exposure. In this study, we examined transcript sequences of monocytic leukemias for evidence of inducible RNA editing. We also examined the role of mitochondrial respiratory inhibition, which induces RNA editing by A3A, in growth of leukemic progenitors of CMML.Methods: Monocytic leukemia samples are obtained from the RPCI Leukemia Bank. Standard diagnostic information included classification (WHO 2008 and FAB), fraction of monocytic component (mature monocytes, promonocytes or monoblasts by flow cytometry and/or aspirate differential count), cytogenetic analyses (conventional karyotype and targeted FISH), and survival data. Leukemic BM mononuclear cells (BM-MNC) prepared by the Ficoll gradient method are thawed and cultured in hypoxia (1% O2) with interferon type 1 (IFN-1). DNA and RNA are isolated. AMoL samples are subjected to nextgen high throughput exome and RNA sequencing. Once variants from DNA and RNA of corresponding samples are obtained, the set difference of variants is extracted to keep those detected on RNA samples. We also examined 3 CMML bone marrow samples for evidence of hypoxia-inducible RNA editing by analyzing selected RNA editing sites. Colony forming unit (CFU) assays are also performed in CMML samples.Results: We find that the A3A gene is robustly expressed inboth in AMoL and CMML. Hypoxia (with or without IFN-1) induced A3A-mediated RNA editing in AMoL and CMML samples. We previously showed that several RNA edited genes by A3A in normal monocytes function in recurrently altered pathways in myeloid leukemias. Sanger sequencing confirmed RNA editing of such genes in monocytic leukemia samples as well (Table 1), suggesting that epitranscriptomic sequence changes may contribute to molecular defects driving leukomogenesis. RNA seq analysis of AMoL samples (n=3) showed that dozens of genes acquire nonsense or missense mutations at the transcript level under the conditions of severe hypoxia and IFN-1. CFU assays of CMML samples showed that inhibition of mitochondrial respiration, which triggers A3A-mediated RNA editing, inhibits the growth of progenitor cells (Figure 1).Conclusions: We show that A3A-mediated RNA editing is active in monocytic leukemias and targets genes functioning in pathways recurrently altered in myeloid leukemias. RNA editing by A3A may contribute to growth arrest and survival of leukemic progenitors under certain stress conditions. To our knowledge, these results provide the first experimental evidence that conditional RNA editing by A3A confers substantial mutational load in myeloid leukemias. Such RNA editing events will dynamically increase the total mutational load in monocytic leukemias, and may create neo-antigens for immune therapy by checkpoint inhibition. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Anti-IL1RAP/CD3 Bispecific Antibody (BsAb) Is a Promising Novel and Effective Therapy for Acute Myeloid Leukemia (AML)

Despite increasing insight of the mechanisms of leukemogenesis, implementation of risk-stratification treatment strategies and use of molecular targeting therapeutics, only a minority of AML patients are cured with chemotherapy-based regimens. Most patients are either primary refractory to induction therapy or subsequently relapse following a brief remission likely due to persistence of chemo-resistant leukemia stem cell (LSC)-enriched subpopulations. Therefore there are urgent unmet therapeutic needs. The principle of CD3 based BsAb therapy is to recruit and activate T cell effector function upon engaging target cells expressing a tumor associated antigen (TAA). Several TAA (e.g.CD33, CD123) have been found to be differentially overexpressed on the surface of AML blasts. Recently, the expression of IL-1 receptor accessory protein (IL1RAP) was reported to be higher on LSCs and more differentiated leukemic progenitor cells (LPCs) than on normal hematopoietic stem cells (HSC), making it an attractive TAA target for immunotherapy. Thus, we hypothesize that an anti-IL1RAP/CD3 BsAb could preferentially target LSCs and LPCs while sparing normal HSCs, thereby offering a window of therapeutic opportunity for AML patients.To test this hypothesis we first determined the expression pattern of IL-1RAP. IL1RAP was differentially expressed on AML cell lines: HL60 and MV4-11 (high), THP-1 (intermediate), and KG-1a (low). Its expression levels were variable on primary blasts from AML patients (n=10). CD34+ peripheral blood mononuclear cells (PBMC) from healthy donors showed little or no IL1RAP expression. Next, we screened 12 anti-IL1RAP monoclonal antibodies from our human phage display antibody library. Based on the results of the BIAcore and antibody dependent cellular cytotoxity (ADCC) assays performed on MV4-11 cells, we selected one lead candidate with binding affinity of ~20nM and EC50 4.9nM for further development. Using knobs-into-holes strategy, we paired this IL1RAP antibody with an anti-CD3 antibody. The dual binding ability of this BsAb was confirmed by flow cytometric analysis. We subsequently proved T cell dependent cytotoxicity (TDCC) of this BsAb using both AML cell lines and AML primary blasts as targets. The anti-IL1RAP/CD3 BsAb induced over 95% of specific killing on all tested AML samples at E: T (Effector : Target) ratio of 1:1 compared with no cytotoxicity effect on the IL-1RAP-negative Raji cells in the 48-hour long term killing (LTK) assay. In line with the cytotoxic data, there was also substantial release of cytokines including IFN-γ, TNF-α and Granzyme B from the AML cells but not Raji cells. TDCC reported over 90% of IL1-RAP specific killing in all but one (only 60%) patient samples (n=10), which had the lowest IL1RAP expression level. The killing of leukemia cells was also observed by time-lapse live cell microscope imaging using luciferase-expressing MV4-11luci+ cells with addition of 100ng/ml BsAb and T cells at E:T ratio of 5:1, which documented effective T cell recruitment and cytolytic activities against MV4-11luci+ cells. We also tested the anti-leukemic activity of this BsAb in vivo using a NSG xenograft mouse model. NSG mice transplanted with 1x106 MV4-11luci+ cells were treated with 200µg anti-IL1RAP/CD3 BsAb plus 3x106 unstimulated T cells, vehicle, T cells alone or anti-CD19/CD3 BsAb (control Ab) plus T cells twice a week for 30 days. In vivo imaging demonstrated effective elimination of the leukemia signal (P<0.001) in MV4-11luci+-engrafted NSG mice treated with T cells and anti-IL1RAP/CD3 BsAb (photon flux (PF) =5.5x105) compared with intense leukemia infiltration in controls treated with either vehicle (PF=3.5x109), T cells alone (PF=4x109), or control anti-CD19/CD3 BsAb plus T cells (PF=2x109). In a separate experiment, treatment with anti-IL1RAP/CD3 BsAb plus T cells of NSG mice engrafted with primary AML blasts led to significant decrease in disease burden than treatment with isotype antibody plusT cells (p=0.004). Further characterization and determination of disease response in these animals is ongoing.In conclusion, we have successfully engineered and characterized an anti-IL1RAP/CD3 BsAb derived from our human naïve phage display library. Our data demonstrated that this BsAb can effectively eradicate AML cells in vitro and in vivo, supporting the clinical translation of this novel immunotherapeutic agent for treatment of AML patients. DisclosuresKwak:InnoLifes: Consultancy, Equity Ownership; Pepromene Bio: Consultancy, Equity Ownership; Celltrion, Inc: Consultancy. Stein:Stemline: Consultancy; Amgen: Consultancy, Speakers Bureau.

Inhibition of Nucleocytoplasmic Export Enhances Selective Elimination of Leukemic Stem Cells in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a hematopoietic stem cell malignancy driven by BCR-ABL1 tyrosine kinase and effectively managed with tyrosine kinase inhibitors (TKIs) such as imatinib. Reactivation of BCR-ABL1 through mutations in the kinase is a common mechanism of resistance, but fails to explain clinical resistance in a significant proportion of cases, a situation referred to as BCR-ABL1-independent resistance. Additionally, most patients require continuous TKI therapy to avoid recurrence of active leukemia, suggesting that primitive, fully leukemogenic CML stem cells (LSCs) are BCR-ABL1-independent. We have previously demonstrated that BCR-ABL1-independent CML cells from patients with overt resistance require functional nucleocytoplasmic export (NCE) and are sensitive to knockdown of the NCE regulator RAN and selective inhibitors of NCE (SINEs) such as the CRM-1 inhibitor KPT-330 (selinexor, Karyopharm) [Khorashad et al. Blood. 2015;125(11):1772-81]. We hypothesize that reliance on NCE may extend to LSCs, the reservoir for persistent CML. To test this, we performed in vivo and in vitro experiments to evaluate the effects of a prototype SINE, KPT-330, on LSCs (defined as CD34+CD38-) and leukemic progenitor cells (LPCs, CD34+CD38+) isolated from newly diagnosed chronic phase CML patients and normal cord blood (CB).Treatment with KPT-330 (50 nM) reduced colony formation by CML (n=4) LPCs and LSCs to 42% and 55% of controls, compared to 78% and 96% for CB (n=3) (Table 1). When combined with imatinib (2.5 µM), KPT-330 treatment dramatically reduced colony formation by LPCs and LSCs to 8% and 2% of controls, compared to 66% and 43% for CB. Single agent KPT-330 (50 nM) had modest effects on apoptosis in LSCs and LPCs, but the combination increased apoptosis in LPCs and LSCs by 280% and 940% relative to controls, compared to 180% and 270% respectively for CB (Table 1). These data suggest that combining KPT-330 with imatinib has the most profound effects on LSCs, with a large differential compared to normal hematopoietic stem cells.To test the effect of KPT-330 on functionally defined LSCs, CML CD34+ cells were cultured ex vivo with or without KPT-330 (50 nM) and/or imatinib (2.5 µM) for 72 hours, followed by plating in long term culture-initiating cell (LTC-IC) assays (n=5) or injection into NSG mice (n=20, 5 per treatment arm). While no reduction in colonies was seen in LTC-IC experiments with single agent KPT-330 or imatinib, a 64% reduction in LTC-IC colonies was observed with combination treatment (p<0.05). Genotyping of LTC-IC colonies by fluorescent-in-situ-hybridization (FISH) revealed that BCR-ABL1+ colonies were reduced by 11% and 22% relative to controls, when treated with KPT-330 or imatinib respectively, but combination treatment reduced BCR-ABL1+ colonies by 38%, significantly lower than either single agent (p<0.05). Thus far results are available from one xenograft experiment. All treatment groups engrafted with a median of 80% human CD45+ cells in bone marrow and 54% in spleen at 12 weeks after injection. Human CD45+ cells were sorted and genotyped. The median proportion BCR-ABL1+ cells in the control was 40%, and was not significantly reduced by imatinib or KPT-330 alone. However combination treatment reduced BCR-ABL+ cells by 42% (p<0.01).To identify potential CRM1 clients regulated in LSCs, we performed a meta-analysis of published gene expression data on CML vs. normal C34+38- or quiescent cells. Candidate genes with differential expression between CML and normal were cross-referenced against putative CRM1 cargos predicted by the LocNES tool [Xu et al. Bioinformatics. 2015;31(9):1357-65]. Confocal microscopy is ongoing to validate cytoplasmic relocalization upon KPT-330 exposure for several candidates, including CD26, an established marker of LSCs that has recently been shown to enhance quiescence in hematopoietic cells [O'Leary et al. Leukemia. 2017 Apr 21;Epub ahead of print].Our data illustrates that interrupting nucleocytoplasmic export by SINEs preferentially eliminates CML LSCs and enhances their sensitivity to TKIs, suggesting that combinations of TKI and SINEs represent a clinical strategy to target BCR-ABL1 independent resistance, as well as persistent residual CML. Mechanistic studies exploring the effects of nuclear entrapment of certain NCE cargo proteins of biological significance in CML LSCs are ongoing and will be reported. [Display omitted] DisclosuresDeininger:Celgene: Research Funding; Incyte: Consultancy; Novartis: Consultancy, Research Funding; ARIAD: Consultancy; Gilead: Research Funding; Pfizer: Consultancy; BMS: Consultancy, Research Funding; Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Leukemic Stem Cell Expression Signatures Identify Novel Therapeutics for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) has a relatively poor 5-year survival rate of 30% in adults, largely due to high rates of relapse, which is thought to be driven by chemoresistant leukemic stem cells (LSCs). A therapy would need to eradicate all LSCs to obtain durable responses in AML patients. Unlike typical cancerous cells, LSCs are often quiescent, allowing them to evade standard therapies and serve as a reservoir for relapse. Thus, the identification of novel anti-LSC therapies could result in the improvement of the clinical outcome of patients suffering from AML.To identify compounds predicted to impede LSC function while not affecting normal hematopoietic stem cells (HSCs), we probed datasets of drug-gene interaction with our previously defined LSC and HSC gene expression signatures generated from 16 primary AML samples and 4 pooled cord blood samples (Eppert et al. Nat Med, 2011), and an additional LSC-signature from 86 AML patients (unpublished data; Ng et al. Nature, 2016). We identified 152 molecules predicted to target LSCs without harming HSCs. Eighty-three of these molecules were screened against a primary AML sample (8227) with a known LSC-containing population (confirmed as CD34+CD38- by xenotransplantation). Viability and phenotype were assessed by flow cytometry. We identified multiple hits in two classes of drugs: 3 drugs in a subclass of steroids and 3 drugs in a specific group of ion pump inhibitors, that preferentially eliminated CD34+CD38- cells over CD15+ blast cells in 8227. Many of these candidates are already in use for other clinical settings. The steroids induced differentiation in the lower nanomolar range (IC50: 0.44-1.30nM), shown by a depletion of primitive CD34+CD38- cells and subsequent expansion of terminally differentiated CD15+ cells. The ion pump inhibitors preferentially targeted the CD34+CD38- cells (IC50 CD34+CD38-: 11.55nM, 24.69nM, 21.78nM) over the CD15+ blast cells (IC50 CD15+: 17.72nM, 32.73nM, 45.09nM). To determine the toxicity of our candidates towards normal stem and progenitor blood cells, we exposed CD34+ enriched human cord blood to the steroids and ion pump inhibitors. All candidates had minimal effects against the CD34+ cord blood cells compared to CD34+ 8227 cells. We then tested the compounds against multiple primary AML samples and observed that at low nanomolar concentrations (0.3-4.0nM) the steroids differentiated AML #9642 (M4e, NPM1-WT, FLT3-WT) but had no effect on AML #9706 (M1, NPM1-WT, FLT3-ITD). For the ion pump inhibitors, AML #184 (M4, NPM1-MT, FLT3-TKD) and AML #9642 were sensitive while AML #116 (M0, NPM1-WT, FLT3-WT) and AML #9706 were more resistant. This variability in sensitivity to both groups of candidates suggests a possible link to subtype or genotype to responsiveness. To evaluate the effect of the steroids and ion pump inhibitors on functional leukemic progenitor cells, 8227 cells were treated with the steroids, ion pump inhibitors or control (DMSO) and then assessed by colony formation unit assay. Treatment with the steroids or ion pump inhibitors resulted in a 2-fold decrease in progenitors (colony formation) compared to DMSO. Next, we will expose a panel of AML patient samples to the candidate compounds in vitro to further elucidate whether there is a link between certain clinical or genotypic features and responsiveness. We will also perform xenotransplantation of treated AML samples into immunodeficient mice to assess the effects on functional LSCs.Overall, we have shown that both the subclass of steroids and specific ion pump inhibitors identified by our bioinformatic approach have anti-AML and anti-LSC properties against particular AML samples in vitro . Future results from our study will provide valuable insight to LSC biology and can lead to new therapeutic approaches for targeting the LSCs, the root of therapy resistance and relapse in AML. DisclosuresNo relevant conflicts of interest to declare.

Targeting WEE1 Kinase after G2/M Checkpoint Arrest Induces Apoptosis in Chronic Myeloid Leukemia Progenitors

BackgroundThe integrity of signaling pathways involved in cell cycle arrest, chromatin remodeling and DNA repair are critical for maintaining the fidelity of replicated DNA. Normal cells repair damaged DNA during G1 arrest; in contrast, leukemic cells often have a deficient G1/S checkpoint and depend on a functional G2/M checkpoint for DNA repair. Aurora Kinase A (AKA) is a key component of centrosome cycle and polar spindle assembly required for regulated progression from G2 to M and throughout M phase. It is frequently overexpressed in cancers, where it correlates with a poor prognosis. Notably, AKA overexpression is associated with genomic instability, one major trait of chronic myeloid leukemia (CML). Our hypothesis is that AKA, together with Polo like kinase 1 (Plk1), cooperates with the constitutive TK activity of the BCR-ABL1 fusion protein by increasing DNA damage, promoting the occurrence of additional genomic alterations and driving TKIs resistance and disease progression to blast crisis.AimsIn this study, the effects of AKA and Plk1 inhibition were evaluated in K562 cells sensitive (K562-S) and resistant (K562-R) to imatinib.MethodsProtein expression and activation was assessed by Western Blotting (WB). Danusertib and Volasertib were used to investigate the effects of AKA and Plk1 inhibition in K562-S and K562-R cells. Both cell lines were incubated with increasing concentrations of Danusertib and Volasertib (0.5-1 µM) or control medium for 24, 48, 72 and 96 hours to perform growth curve experiments and to identify the best sub-lethal drug doses in liquid medium. The percentage of apoptotic cells was quantified by annexin V/propidium iodide staining and flow cytometry. WB experiments were performed to evaluate AKA and Plk1 expression and activation before and after treatment with Danusertib and Volasertib alone or in combination with the Wee1 inhibitor MK1775. Further experiments were performed using polyclonal antibodies against cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 to confirm apoptosis activation. PARP cleavage and γH2AX expression and activation were also tested. The effects of Danusertib and Volasertib on cell cycle progression were evaluated by flow cytometry in K562-S and K562-R cells after incubation in medium containing 0.5 µM of each drugs for 24 h.ResultsBoth Danusertib and Volasertib (0.5 µM for 24h) were effective in cell growth inhibition in K562-S and K562-R by inducing cells to undergo cell cycle arrest and apoptosis. Moreover, they caused a dose- and time-dependent reduction of the G0/G1 cell fraction and an increase of the G2/M fraction. Cell cycle arrest was associated with increased levels of phospho (p)-Chk1 and p-Chk2, p-cyclin B1, p-cdc2 and p-Wee1. Using a Wee1 inhibitor (MK1775) after 24h treatment with Danusertib and Volasertib 0.5 µM, when cells were arrested in G2 phase and Wee1 was overexpressed and hyper-activated, resulted in a synergistic inhibition of cell viability in both K562-S and -R (Figure 1). MK1775 combined with either Danusertib or Volasertib caused a time-dependent increase of annexin-V-positive cells by activating the mitochondrial apoptotic pathway as reflected by an increment of Bax expression and induction of the cleavage of caspase-3, -8 and -9 and PARP. Moreover, both drug combinations induced a significant increase of the DNA double-strand break marker γH2AX, suggesting that Wee1 inhibition promotes mitosis and propagates genomic instability by forcing the cells through successive replication cycles, ultimately resulting in apoptosis from mitotic catastrophe.ConclusionsOur results provide support for MK1775 to be used in synergistic combination therapies to improve the outcomes of patients with CML in blast crisis or resistant to multiple lines of TKI therapy.Supported by ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. [Display omitted] DisclosuresCastagnetti:Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Gugliotta:Incyte: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Rosti:Incyte: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Martinelli:Johnson&Johnson: Consultancy; Roche: Consultancy; Ariad/Incyte: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen: Consultancy. Soverini:Bristol-Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy.

MLKL promotes cellular differentiation in myeloid leukemia by facilitating the release of G-CSF

The blockade of cellular differentiation represents a hallmark of acute myeloid leukemia (AML), which is largely attributed to the dysfunction of lineage-specific transcription factors controlling cellular differentiation. However, alternative mechanisms of cellular differentiation programs in AML remain largely unexplored. Here we report that mixed lineage kinase domain-like protein (MLKL) contributes to the cellular differentiation of transformed hematopoietic progenitor cells in AML. Using gene-targeted mice, we show that MLKL facilitates the release of granulocyte colony-stimulating factor (G-CSF) by controlling membrane permeabilization in leukemic cells. Mlkl−/− hematopoietic stem and progenitor cells released reduced amounts of G-CSF while retaining their capacity for CSF3 (G-CSF) mRNA expression, G-CSF protein translation, and G-CSF receptor signaling. MLKL associates with early endosomes and controls G-CSF release from intracellular storage by plasma membrane pore formation, whereas cell death remained unaffected by loss of MLKL. Of note, MLKL expression was significantly reduced in AML patients, specifically in those with a poor-risk AML subtype. Our data provide evidence that MLKL controls myeloid differentiation in AML by controlling the release of G-CSF from leukemic progenitor cells.

Towards precision medicine for AML.

With rapid advances in sequencing technologies, tremendous progress has been made in understanding the molecular pathogenesis of acute myeloid leukaemia (AML), thus revealing enormous genetic and clonal heterogeneity, and paving the way for precision medicine approaches. The successful development of precision medicine for patients with AML has been exemplified by the introduction of targeted FLT3, IDH1/IDH2 and BCL-2 inhibitors. When used as single agents, these inhibitors display moderate antileukaemic activity. However, augmented clinical activity has been demonstrated when they are administered in combination with drugs with broader mechanisms of action targeting epigenetic and/or other oncogenic signalling pathways or with conventional cytotoxic agents. The development of immunotherapies has been hampered by the expression of antigens that are expressed by both leukaemic and non-malignant haematopoietic progenitor cells; nonetheless, a diverse range of immunotherapies are now entering clinical development. This myriad of emerging agents also creates challenges, such as how to safely combine agents with different mechanisms of action, the need to circumvent primary and secondary resistance, and new challenges in future clinical trial design. In this Review, we discuss the current state of precision medicine for AML, including both the potential to improve patient outcomes and the related challenges.

Inhibitory effects of Tomivosertib in acute myeloid leukemia.

The MAPK-interacting kinases 1 and 2 (MNK1/2) have generated increasing interest as therapeutic targets for acute myeloid leukemia (AML). We evaluated the therapeutic potential of the highly-selective MNK1/2 inhibitor Tomivosertib on AML cells. Tomivosertib was highly effective at blocking eIF4E phosphorylation on serine 209 in AML cells. Such inhibitory effects correlated with dose-dependent suppression of cellular viability and leukemic progenitor colony formation. Moreover, combination of Tomivosertib and Venetoclax resulted in synergistic anti-leukemic responses in AML cell lines. Mass spectrometry studies identified novel putative MNK1/2 interactors, while in parallel studies we demonstrated that MNK2 - RAPTOR - mTOR complexes are not disrupted by Tomivosertib. Overall, these findings demonstrate that Tomivosertib exhibits potent anti-leukemic properties on AML cells and support the development of clinical translational efforts involving the use of this drug, alone or in combination with other therapies for the treatment of AML.

Leukemic Cytokines co-opt Regulatory T cell and Th17 immunity to promote chronic myeloid leukemia

Abstract Non-immune roles of Regulatory T cells (Tregs) have been described across various tissues. However, the mechanisms by which T cells regulate the bone marrow in myeloproliferative neoplasms lack resolution. A hallmark of leukemic progression is the increase in pro-inflammatory cytokines, cellular inflammation, and an expansion of CD4 T cells. In this study, we demonstrate that this inflammatory milieu promotes pathogenic skewing of both regulatory and effector T cells. We characterized the altered CD4+ T cell compartment in chronic myeloid leukemia (CML) and we found that Tregs are unstable and dysregulated across leukemic tissues. Specifically, the CML environment effectively co-opts Treg function and stability. This contributes to a phenotype of immune activation as well as an increase of pathogenic inflammatory T cells. Notably, our findings reveal that Treg/Th17 ratios are disrupted in leukemia and that loss of Tregs and activation of Th17 cells influence disease progression. Additionally, we found that Treg-secreted IL-10 directly influences myeloid and neutrophil differentiation of leukemic progenitors has beneficial anti-inflammatory effects. We demonstrate that restoring Treg function and expanding their IL-10 production can be used therapeutically to limit leukemic progression. Our results support the hypothesis that Tregs are compromised during leukemic progression as a consequence of pro-inflammatory cytokines from the myeloid associated disease. These findings highlight how the Treg/Th17 balance regulates disease progression. Furthermore, these studies provide an avenue by which Treg secreted IL-10 can be used for therapeutic intervention in the context of hematopoietic malignancies.

Taming Cell-to-Cell Heterogeneity in Acute Myeloid Leukaemia With Machine Learning.

Acute Myeloid Leukaemia (AML) is a phenotypically and genetically heterogenous blood cancer characterised by very poor prognosis, with disease relapse being the primary cause of treatment failure. AML heterogeneity arise from different genetic and non-genetic sources, including its proposed hierarchical structure, with leukemic stem cells (LSCs) and progenitors giving origin to a variety of more mature leukemic subsets. Recent advances in single-cell molecular and phenotypic profiling have highlighted the intra and inter-patient heterogeneous nature of AML, which has so far limited the success of cell-based immunotherapy approaches against single targets. Machine Learning (ML) can be uniquely used to find non-trivial patterns from high-dimensional datasets and identify rare sub-populations. Here we review some recent ML tools that applied to single-cell data could help disentangle cell heterogeneity in AML by identifying distinct core molecular signatures of leukemic cell subsets. We discuss the advantages and limitations of unsupervised and supervised ML approaches to cluster and classify cell populations in AML, for the identification of biomarkers and the design of personalised therapies.

Targeting AXL kinase sensitizes leukemic stem and progenitor cells to venetoclax treatment in acute myeloid leukemia

AbstractThe abundance of genetic abnormalities and phenotypic heterogeneities in acute myeloid leukemia (AML) poses significant challenges to the development of improved treatments. Here, we demonstrated that a key growth arrest-specific gene 6/AXL axis is highly activated in cells from patients with AML, particularly in stem/progenitor cells. We developed a potent selective AXL inhibitor that has favorable pharmaceutical properties and efficacy against preclinical patient-derived xenotransplantation (PDX) models of AML. Importantly, inhibition of AXL sensitized AML stem/progenitor cells to venetoclax treatment, with strong synergistic effects in vitro and in PDX models. Mechanistically, single-cell RNA-sequencing and functional validation studies uncovered that AXL inhibition, alone or in combination with venetoclax, potentially targets intrinsic metabolic vulnerabilities of AML stem/progenitor cells and shows a distinct transcriptomic profile and inhibits mitochondrial oxidative phosphorylation. Inhibition of AXL or BCL-2 also differentially targets key signaling proteins to synergize in leukemic cell killing. These findings have a direct translational impact on the treatment of AML and other cancers with high AXL activity.

Phosphorylation state of the histone variant H2A.X controls human stem and progenitor cell fate decisions

Histone variants (HVs) are a subfamily of epigenetic regulators implicated in embryonic development, but their role in human stem cell fate remains unclear. Here, we reveal that the phosphorylation state of the HV H2A.X (γH2A.X) regulates self-renewal and differentiation of human pluripotent stem cells (hPSCs) and leukemic progenitors. As demonstrated by CRISPR-Cas deletion, H2A.X is essential in maintaining normal hPSC behavior. However, reduced levels of γH2A.X enhances hPSC differentiation toward the hematopoietic lineage with concomitant inhibition of neural development. In contrast, activation and sustained levels of phosphorylated H2A.X enhance hPSC neural fate while suppressing hematopoiesis. This controlled lineage bias correlates to occupancy of γH2A.X at genomic loci associated with ectoderm versus mesoderm specification. Finally, drug modulation of H2A.X phosphorylation overcomes differentiation block of patient-derived leukemic progenitors. Our study demonstrates HVs may serve to regulate pluripotent cell fate and that this biology could be extended to somatic cancer stem cell control.

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In This Issue

Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

SummaryThe aberrant expression of dopamine receptors (DRDs) in acute myeloid leukemia (AML) cells has encouraged the repurposing of DRD antagonists such as thioridazine (TDZ) as anti-leukemic agents. Here, we access patient cells from a Phase I dose escalation trial to resolve the cellular and molecular bases of response to TDZ, and we extend these findings to an additional independent cohort of AML patient samples tested preclinically. We reveal that in DRD2+ AML patients, DRD signaling in leukemic progenitors provides leukemia-exclusive networks of sensitivity that spare healthy hematopoiesis. AML progenitor cell suppression can be increased by the isolation of the positive enantiomer from the racemic TDZ mixture (TDZ+), and this is accompanied by reduced cardiac liability. Our study indicates that the development of DRD-directed therapies provides a targeting strategy for a subset of AML patients and potentially other cancers that acquire DRD expression upon transformation from healthy tissue.

A robust approach for the generation of functional hematopoietic progenitor cell lines to model leukemic transformation.

Studies of molecular mechanisms of hematopoiesis and leukemogenesis are hampered by the unavailability of progenitor cell lines that accurately mimic the situation in vivo. We now report a robust method to generate and maintain LSK (Lin-, Sca-1+, c-Kit+) cells, which closely resemble MPP1 cells. HPCLSKs reconstitute hematopoiesis in lethally irradiated recipient mice over >8 months. Upon transformation with different oncogenes including BCR/ABL, FLT3-ITD, or MLL-AF9, their leukemic counterparts maintain stem cell properties in vitro and recapitulate leukemia formation in vivo. The method to generate HPCLSKs can be applied to transgenic mice, and we illustrate it for CDK6-deficient animals. Upon BCR/ABLp210 transformation, HPCLSKsCdk6-/- induce disease with a significantly enhanced latency and reduced incidence, showing the importance of CDK6 in leukemia formation. Studies of the CDK6 transcriptome in murine HPCLSK and human BCR/ABL+ cells have verified that certain pathways depend on CDK6 and have uncovered a novel CDK6-dependent signature, suggesting a role for CDK6 in leukemic progenitor cell homing. Loss of CDK6 may thus lead to a defect in homing. The HPCLSK system represents a unique tool for combined in vitro and in vivo studies and enables the production of large quantities of genetically modifiable hematopoietic or leukemic stem/progenitor cells.

IGFBP7 activates retinoid acid-induced responses in acute myeloid leukemia stem and progenitor cells.

Treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) in combination with low doses of arsenic trioxide or chemotherapy leads to exceptionally high cure rates (>90%). ATRA forces APL cells into differentiation and cell death. Unfortunately, ATRA-based therapy has not been effective among any other acute myeloid leukemia (AML) subtype, and long-term survival rates remain unacceptably low; only 30% of AML patients survive 5 years after diagnosis. Here, we identified insulin-like growth factor binding protein 7 (IGFBP7) as part of ATRA-induced responses in APL cells. Most importantly, we observed that addition of recombinant human IGFBP7 (rhIGFBP7) increased ATRA-driven responses in a subset of non-APL AML samples: those with high RARA expression. In nonpromyelocytic AML, rhIGFBP7 treatment induced a transcriptional program that sensitized AML cells for ATRA-induced differentiation, cell death, and inhibition of leukemic stem/progenitor cell survival. Furthermore, the engraftment of primary AML in mice was significantly reduced following treatment with the combination of rhIGFBP7 and ATRA. Mechanistically, we showed that the synergism of ATRA and rhIGFBP7 is due, at least in part, to reduction of the transcription factor GFI1. Together, these results suggest a potential clinical utility of IGFBP7 and ATRA combination treatment to eliminate primary AML (leukemic stem/progenitor) cells and reduce relapse in AML patients.

Diphtheria Toxin Fused to Granulocyte-Macrophage Colony-Stimulating Factor Is Toxic to Blasts From Patients With Juvenile Myelomonocytic Leukemia and Chronic Myelomonocytic Leukemia

AbstractWe have previously demonstrated that human granulocyte-macrophage colony-stimulating factor fused to a truncated diphtheria toxin (DT388-GM-CSF) is toxic to patient acute myeloid leukemia progenitors bearing the GM-CSF receptor, but not normal marrow progenitors. We now report that exposure of mononuclear cells from five of seven (71%) juvenile myelomonocytic leukemia (JMML) patients and from 12 of 20 (60%) adult chronic myelomonocytic leukemia (CMML) patients to 10-9 mol/L DT388-GM-CSF for 48 hours in culture reduces the number of cells capable of forming colonies in semisolid medium (colony-forming units–leukemia) 10-fold to 300-fold (1 to 2.5 log decrease). In contrast, normal myeloid progenitors (colony-forming unit–granulocyte-macrophage) from six different donors treated and assayed under identical conditions were consistently insensitive to the same fusion toxin even when treated as highly purified CD34+ cells. The leukemic progenitors from the two other JMML patients showed intermediate sensitivity to DT388-GM-CSF and the leukemic progenitors from eight of the 20 (40%) CMML patients were not different from normal progenitors. Parallel measurements of the number and affinity of GM-CSF receptors on cells from the same samples showed no consistent differences between JMML, CMML, and normal light density or CD34+ bone marrow cells. The increased sensitivity of leukemic progenitors from all JMML progenitors and some CMML patients to the fusion toxin is therefore not likely to be explained by an increased density of GM-CSF receptors on these cells. We also examined the DT388-GM-CSF sensitivity of two murine cell lines transfected with cDNAs encoding varying portions of the human GM-CSF receptor α and/or β chains. These studies showed that high-affinity ligand binding was sufficient for DT388-GM-CSF–induced toxicity, as this could occur even in the absence of functional signal transduction and that the background of the host cell had a major influence on the degree to which this decreased the toxicity of DT388-GM-CSF. The selective sensitivity to DT388-GM-CSF of leukemic progenitors from a majority of JMML and CMML patients suggests that this agent could have therapeutic potential for some patients with these diseases.

Malignant Progenitors From Patients With Acute Myelogenous Leukemia Are Sensitive to a Diphtheria Toxin–Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein

AbstractWe have previously demonstrated that human granulocyte-macrophage colony-stimulating factor (GM-CSF) fused to a truncated diphtheria toxin (DT388-GMCSF) kills acute myelogenous leukemia (AML) cell lines bearing the GM-CSF receptor. We now report that exposure of malignant cells from 50 different patients with AML for 48 hours in culture to DT388-GMCSF reduces by a median of 1.6 logs (range, 0 to 3.7 logs) the number of leukemic cells capable of forming colonies in semisolid media (leukemic colony-forming cells [CFU-L]) with a median IC50 of 3 × 10−12 mol/L (range, 5 to >4,000 × 10−12 mol/L). Furthermore, the cell kill is dependent on the presence of high-affinity GM-CSF receptors on leukemic blasts, because CFU-L from 27 of 28 AML samples expressing ≥35 GM-CSF receptors per cell were inhibited by the toxin, whereas the colony growth from all 4 leukemic samples (2 AML, 1 acute lymphoblastic leukemia [ALL], and 1 prolymphocytic leukemia [PLL]) that had less than 35 receptors per cell was unaffected by the drug. Sensitivity of CFU-L to DT388-GMCSF was seen regardless of the clinical responsiveness of the patient's leukemia to standard chemotherapy agents. In contrast, clonogenic cells from normal bone marrow formed colonies at near control numbers after exposure to much higher toxin concentrations (4 × 10−9 mol/L) than those required to kill CFU-L from most patients. Thus, leukemic progenitors isolated directly from the peripheral blood of most AML patients show the same sensitivity to DT388-GMCSF as previously demonstrated for AML cell lines. Under the same conditions of exposure, normal hematopoietic progenitors are relatively unaffected by DT388-GMCSF, suggesting its potential as a therapeutic agent in AML.

Mir142 loss unlocks IDH2R140-dependent leukemogenesis through antagonistic regulation of HOX genes

AML is a genetically heterogeneous disease and understanding how different co-occurring mutations cooperate to drive leukemogenesis will be crucial for improving diagnostic and therapeutic options for patients. MIR142 mutations have been recurrently detected in IDH-mutated AML samples. Here, we have used a mouse model to investigate the interaction between these two mutations and demonstrate a striking synergy between Mir142 loss-of-function and IDH2R140Q, with only recipients of double mutant cells succumbing to leukemia. Transcriptomic analysis of the non-leukemic single and leukemic double mutant progenitors, isolated from these mice, suggested a novel mechanism of cooperation whereby Mir142 loss-of-function counteracts aberrant silencing of Hoxa cluster genes by IDH2R140Q. Our analysis suggests that IDH2R140Q is an incoherent oncogene, with both positive and negative impacts on leukemogenesis, which requires the action of cooperating mutations to alleviate repression of Hoxa genes in order to advance to leukemia. This model, therefore, provides a compelling rationale for understanding how different mutations cooperate to drive leukemogenesis and the context-dependent effects of oncogenic mutations.

Transcriptional Heterogeneity Governs Cell Fate Diversification during Pre-Leukemia to Leukemia Progression

Acute myeloid leukemia (AML) is a heterogenous clonal disorder of hematopoietic progenitor cells with a dismal survival. It has a strong reliance on epigenetic and transcriptional factors for disease progression. Accordingly, we have previously identified KAT2A, a histone acetyl-transferase, as a requirement for AML maintenance; where chemical inhibition of KAT2A promotes differentiation of AML cell lines (Tzelepis et al., 2016, Cell Reports 17, 1193-1205). More recently, using a conditional knockout mouse model for Kat2a we showed that it sustains KMT2A/MLLT3 AML stem cells. Kat2a is a classical regulator of transcriptional variability, it's loss leads to cell-to-cell heterogeneity in transcription levels specifically from genes involved in ribosomal biogenesis and translation (Domingues et al., 2020, eLife 9:e51754). No recurrent mutations in the KAT2A gene have been described in AML, and it is unclear if and how it participates in pre-leukemia-to-AML progression. Herein, we use our conditional Kat2a knockout mouse model to analyze the effects of Kat2a loss in biology of RUNX1-RUNX1T1(9a) and Idh1R132H-initiated AML. These models represent forms of human disease with a prolonged pre-leukemia phase that typically require additional mutations for leukemia progression. We observed that loss of Kat2a accelerates leukemia initiation and progression in vivo. This acceleration was a consequence of fixation of transformed Kat2a KO cells in vivo which reflects as enhanced self-renewal capacity in vitro as measured by serial re-plating colony forming assay. Given the central role of Kat2a in limiting cell-to-cell transcription heterogeneity, we interrogated a potential link between loss of Kat2a, its consequent increase in transcriptional heterogeneity and pre-leukemia progression. For this, we performed single-cell RNA sequencing (scRNA-seq) of early-stage Kat2a WT and Kat2a KO RUNX1-RUNX1T1(9a) pre-leukaemia. Compatible with our previous observation, we observed that Kat2a KO cells were more heterogenous transcriptionally. Interestingly, this was accompanied by diversification of cell fates towards B-lymphocytes and monocytes. Furthermore, pseudo-temporal ordering of single Kat2a KO cells revealed highly branched trajectory heavily populated with intermediate stages of transformation; including accumulation of leukemia progenitors with RUNX1-RUNX1T1 signature. In contrast, Kat2a WT cells have linear normal hematopoiesis trajectory with minimal branching and an abrupt transition towards candidate leukemia progenitor state. Pathway analysis of Kat2a KO leukemia progenitor cells indicated perturbation of ribosomal biogenesis and translation associated genes. In order to test how these changes contributed to transformation, we performed S6K1 inhibition on Kat2a WT cells which transiently promoted transformation in vitro in both RUNX1-RUNX1T1(9a) and Idh1R132H cells, thus, phenocopying the effects of Kat2a loss. This suggested a mechanistic contribution of observed transcriptional changes in protein synthesis machinery towards leukemia progression. Taken together, our work suggests that loss of Kat2a results in diversification of cell fates, including with increased accessibility to cell states prone to transformation. Furthermore, these cells, prone to transformation, may benefit from a low biosynthetic activity that promotes their progression to leukemia state. We hypothesize that Kat2a loss may function similarly in the context of other malignancies. In the future, this knowledge may aid in development of early diagnostic tools and suggest bespoke therapeutic interventions. Figure Disclosures Prabakaran: Noncodomics: Consultancy. Vassiliou:Kymab Ltd - Monoclonal antibody company. Currently not working in myeloid cancers or clonal haematopoiesis.: Consultancy.