FLT3-mutated acute myeloid leukemia (AML) remains difficult to treat due to frequent resistance to FLT3 inhibitors like midostaurin. In this study, we observed a progenitor-like CD38+CD45RA+ leukemic cell population that may be associated with midostaurin resistance. Midostaurin-resistant cells display disrupted membrane architecture and a shift in signaling from STAT5 to PI3K/AKT, favoring survival over apoptosis. Functional drug testing was consistent with clinical response to midostaurin, and together with multi-omic profiling, including single-cell and proteomic analyses, indicated the presence and relevance of this resistant phenotype. Drug combination screening revealed that co-targeting with SMAC mimetics restores apoptotic competence and selectively depletes the resistant population when combined with midostaurin. In contrast, venetoclax combinations preferentially affected CD34hi cells, underscoring distinct subpopulation vulnerabilities. These findings may point to a biologically relevant mechanism underlying midostaurin resistance.

Acute myeloid leukaemia (AML) is an aggressive blood cancer characterized by the unregulated proliferation of immature myeloblasts. Gene mutations have been shown to have a large effect on pathogenesis, inter-tumour heterogeneity and clinical outcomes in AML1-8; however, the role of epigenetic alterations in these respects has been investigated less extensively. Here we use ATAC-seq (assay for transposase-accessible chromatin with sequencing) in a cohort of 1,563 individuals with a recent diagnosis of AML (the 'eCHROMA' cohort) to show that AML can be classified into 16 subgroups on the basis of chromatin accessibility profiles. Multiomics analyses of gene mutations, the transcriptome, DNA methylation and histone marks show that these ATAC subgroups exhibit distinct driver mutations, differentiation states, gene expression, DNA methylation and super-enhancer profiles, and are also associated with clinical outcomes. These findings were validated in independent cohorts. Single-cell ATAC sequencing reveals that all leukaemic cells in each subgroup share a common chromatin accessibility profile, which suggests that subgroup-specific epigenomic fingerprints underlie the ATAC-based classification. Mechanistically, the subgroups have distinct gene-regulatory networks that are driven by the activities of key transcription factors in haematopoiesis, and in which subgroup-specific super-enhancers have a pivotal role. Multiomics single-cell analysis further reveals deregulated trajectories of differentiation coupled with chromatin accessibility and gene expression. Notably, ATAC subgroups have an independent prognostic effect, compared with genomic classification, and are associated with particular drug sensitivities. In summary, ATAC-based chromatin profiling, combined with multiomics data, provides insights into AML pathogenesis beyond genomics and constitutes a valuable resource for AML research.

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML), characterized by a fusion between the PML and RARA genes and by a block in the myeloid maturation at the promyelocytic stage. This study investigates the epigenetic landscape of APL by integrating ChIP-seq data on eight histone modifications and RNA-seq in APL as well as non-APL AML. APL showed a distinct chromatin profile that differed from non-APL AML. We describe APL-specific changes in H3K27ac, H3K9me3, and H3K27me3 with impact on enhancer activity, repression of transposable elements, and Polycomb regulated gene repression. The APL-specific H3K27ac pattern identifies APL-specific enhancer and super-enhancer regions, including a subset of enhancers that are bound by the PML-RARA fusion protein. While chromatin bound specifically by PML-RARA were dominantly active, APL was also characterized by gain of APL-specific heterochromatin states with significant gains of H3K9me3 enriched lamina-associated domains and the transposable elements LINE, LTR, and SINE. These findings suggest a unique enhancer and heterochromatin profile in APL, with implications for transcription regulation and treatment response. These findings offer novel insights into the pathogenesis of APL.

Abstract Background Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous disease. While traditional classification systems such as FAB relied on morphology and immunophenotype, recent classifications (WHO, ICC) emphasize genetic alterations for diagnosis and therapy. However, more directly involved in the phenotypic cellular inheritance, the epigenomic profile may offer an additional dimension for understanding AML heterogeneity. Previously, we performed ATAC-seq of 1,536 AML samples and identified 16 epigenetically defined AML subgroups (subgroups A-P), which were associated with distinct clinical, genetic, and transcriptional features (Ochi et al., ASH 2024). In the present study, we extend these findings by using single-cell RNA and ATAC-seq (scRNA/ATAC-seq) profiling to explore intra- and inter-tumor epigenetic heterogeneity, transcriptional regulation, and hierarchical differentiation trajectories across AML subgroups. Methods We performed multi-platform scRNA/ATAC-seq on 36 AML samples including all the 16 epigenomic subgroups and 4 remission samples (as controls), profiling a total of 281,167 mononuclear cells. Computational analyses included cell clustering, projection onto normal hematopoiesis reference maps, pseudotime inference, transcription factor (TF) activity analysis using SCENIC+, and leukemic stem cell (LSC) signature scoring. Results scATAC-based clustering showed that leukemic cells from individual AML samples formed distinct clusters largely separated from normal cells, typically co-clustered by subgroup, regardless of their differentiation status, indicating that leukemic cells within each subgroup share a distinct intrinsic epigenetic program. Projection onto a normal hematopoiesis reference map revealed divergent differentiation arrest among subgroups, even within genetically similar cases. For instance, all NPM1-mutant subgroups (D–F) were HOX-related but differed in differentiation states: subgroup E was arrested at the HSC stage, D at the GMP stage, and F contained both progenitors and mature cells. Single-cell analysis also refined differentiation states of subgroups F-H: while bulk ATAC-based deconvolution suggested monocyte enrichment in these subgroups, single-cell analysis revealed mature monocytes predominated in H, whereas F and G were enriched for immature promonocytes. Combined with LSC signature analysis based on gene expression, pseudotime analysis demonstrated that cells showing high LSC scores consistently mapped to early stages of the differentiation trajectory across all subgroups. These results indicate the presence of a conserved leukemic hierarchy, with stem-like cells positioned at the apex, irrespective of epigenetic subgroup, thereby underpinning AML pathogenesis. We further analyzed TF activity using the SCENIC+ program based on scRNA/ATAC-seq data, which identified key TFs enriched in each subgroup, many of which overlapped with those found in bulk RNA/ATAC-seq analysis. Pseudotime analysis further showed that TFs exhibited subgroup-specific activation dynamics. For example, HOXA9 was activated throughout the myeloid differentiation trajectory in HOX-related subgroups but peaked at different stages according to subgroups. In the RUNX1-enriched subgroup, IRF8 and BCL11A were both active but peaked at mature and immature stages, respectively. These findings indicate that key TFs exhibit subgroup-specific activation patterns at distinct stages along the myeloid differentiation trajectory. Conclusion Single-cell multi-omics analysis reveals that AML epigenetic subgroups exhibit distinct but hierarchically organized differentiation trajectories, driven by dynamically regulated TF programs. These findings refine our understanding of AML diversity and may inform more precise, differentiation-stage–specific therapeutic strategies.

As the non-coding genome remains poorly characterized in acute myeloid leukemia (AML), we aimed to identify and functionally characterize novel long non-coding RNAs (lncRNAs) relevant to AML biology and treatment. We first identified lncRNAs overexpressed in AML blasts and, among them, discovered a novel transcript, which we named myeloid and AML-associated intergenic long non-coding RNA (MALNC). MALNC is overexpressed in AML, particularly in cases with the PML-RARA fusion or IDH2R140/NPM1 co-mutations, and is associated with a distinct gene expression profile. Functional studies showed that MALNC knockout impairs AML cell proliferation and colony formation, enhances ATRA-induced differentiation, and sensitizes cells to arsenic trioxide. Transcriptomic analysis revealed that MALNC loss alters the expression of retinoic acid pathway genes, and chromatin binding studies showed that MALNC binds to genes related to the retinoic acid and Rho GTPase pathways. In conclusion, we have identified MALNC as a novel lncRNA that promotes leukemic cell proliferation, counteracts ATRA-induced differentiation, and modulates drug sensitivity in AML.

Cancer drug resistance remains a critical challenge in cancer therapy, necessitating advanced approaches to understand and counteract this phenomenon. Acute myeloid leukemia (AML), an aggressive and heterogeneous cancer, often exhibits resistance to chemotherapy, contributing to high relapse rates. We are employing a Cellular Thermal Shift Assay (CETSA)-driven targeted proteomics workflow to investigate drug response biomarkers and resistance mechanisms, with the ultimate goal of enabling personalized AML therapies. In our previous publication (Ramos et al., 2024) we identified several biomarkers induced by different cancer drugs. CETSA allows for the direct assessment of a diverse range of biochemical effects in cells, providing crucial insights into drug-induced biomarker responses. Initial results from our study reveal distinct CETSA differences between drug treatments and inter-patient variability in CETSA biomarkers, even for the same treatment. For example, while some patients show a robust response to venetoclax, others exhibit limited biomarker changes. Limited biomarker response to venetoclax and no detectable CETSA apoptosis biomarker response to azacitidine were observed in one patient, with azacitidine-specific changes, suggesting potential resistance downstream of these markers. Additionally, some patients appear highly responsive to venetoclax but not to azacitidine, with no additive effect observed when the two drugs are combined. These findings underscore the variability in therapeutic response across patients and emphasize the need for personalized approaches in AML management. By quantifying protein stability levels following treatment with apoptosis-inducing drugs, this targeted approach confirms the relevance of previously identified biomarkers and supports their role in predicting therapeutic efficacy. Although we do not yet have data from relapsed patients, obtaining such samples is a key goal for the future. These data will allow us to evaluate whether initial treatment responses align with relapse outcomes and may provide critical insights into resistance mechanisms. Understanding CETSA proteomic changes in relapsed patients could uncover key biochemical shifts associated with treatment resistance, ultimately guiding the development of more effective and personalized therapeutic strategies. While this study is ongoing and not yet influencing clinical practice, the emerging data supports the potential for application of CETSA in personalized biomarker-driven cancer therapy. By measuring CETSA-derived biomarkers with targeted proteomics, we aim to lay the foundation for real-time, precision-guided treatment in AML. Furthermore, these insights may extend to other cancer types, advancing the broader goal of personalized oncology. - Ramos et al. Proteome-wide CETSA reveals diverse apoptosis-inducing mechanisms converging on an initial apoptosis effector stage focused at the peripheral region of the nucleus. Cell Reports, 2;43(10):114784. Anderson Daniel Ramos, Smaranda Bacanu, Sara Lööf, Sofia Bengtzén, Jiawen Lyu, Sören Lehmann, Pär Nordlund. CETSA-driven targeted proteomics for personalized AML therapies: Investigating drug response biomarkers and resistance mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB198.

Cytarabine (ara-C) and fludarabine (F-ara-A) are key drugs in leukaemia treatment. SAMHD1 is known to confer resistance to ara-C and F-ara-A, and we previously identified ribonucleotide reductase inhibitors as indirect SAMHD1 inhibitors in a phenotypic screen. The inosine monophosphate dehydrogenase (IMPDH) inhibitor mycophenolic acid (MPA) was also a hit in this screen. IMPDH inhibitors (IMPDHi) have previously shown efficacy against KMT2A-rearranged (KMT2Ar) acute myeloid leukaemia (AML). We investigated whether IMPDH inhibition could enhance the effect of ara-C and F-ara-A in AML cell lines and primary AML samples, and whether this effect was linked to KMT2A status. We found that sensitivity to IMPDHi was independent of KMT2A status. IMPDHi synergized with ara-C and F-ara-A in a SAMHD1-dependent manner in a subset of AML cells, but not in acute lymphoblastic leukaemia cell lines. Mechanistically, IMPDHi depleted allosteric SAMHD1 activators GTP and dGTP, thereby increasing active triphosphate metabolites in SAMHD1-proficient, but not SAMHD1-deficient, cells. Our findings suggest that the addition of IMPDHi to ara-C and F-ara-A may have therapeutic benefits in some AML cases.

Despite intensive treatment, patients with relapsed or refractory acute myeloid leukaemia (AML) have a dismal prognosis. A cornerstone therapy for relapsed/refractory AML is a combination of fludarabine (F-ara-A) and cytarabine (ara-C), so-called FLA. As the enzyme SAMHD1 mediates resistance to both ara-C and F-ara-A, we investigated whether SAMHD1 inhibition via hydroxyurea (hydroxycarbamide; HU) could improve FLA efficacy. Here, we show that HU synergistically enhanced ara-C-, F-ara-A- and FLA-induced cytotoxicity in an SAMHD1-dependent manner in AML cell lines, primary AML cells and an immunocompetent AML mouse model. Mechanistically, HU significantly increased the active metabolite triphosphates of ara-C and F-ara-A in FLA combinations. Furthermore, leukaemic SAMHD1 protein expression negatively correlated with overall survival in a cohort of FLA-treated refractory AML patients. Our findings suggest that the addition of HU improves the efficacy of FLA-based regimens and warrant clinical trials to test the safety and efficacy of this combination in patients with relapsed/refractory AML.

Background Acute myeloid leukemia (AML) is a heterogeneous disease and is primarily defined by genetic abnormalities. Although accumulating evidence suggests the role of epigenetics in the pathogenesis of AML, it has not fully been investigated in a large cohort of patients. Methods We enrolled 1,563 primary AML cases from Swedish (n=1,040) and Japanese (n=523) cohorts and performed ATAC-seq (n=1,563) as well as multi-omics analysis, including targeted-capture sequencing (n=1,563), RNA-seq (n=1,398), whole-genome sequencing (n=207), ChIP-seq (n=120), drug screening (n=112), and single-cell RNA/ATAC-seq (n=31). Results ATAC-seq analysis identified 185K recurrent peaks, most of which were found within intergenic/intronic regions. An unbiased clustering analysis based on ATAC-seq identified 16 unique subgroups with distinct genetic drivers, transcriptome profiles, differentiation states, key transcription factors, and clinical features. Among these, three groups were well-known entities defined by t(8;21), inv(16), and t(15;17). In contrast, the remaining 13 subtypes represent a novel classification framework not defined by single genomic abnormalities. Patients with HOX-related gene abnormalities, such as NPM1 mutation as well as KMT2A and NUP98 rearrangement, converged into four subgroups (D-G). Associated with global chromatin changes in the HOXA locus from repressive to active state, these ATAC subgroups were characterized in common by an elevated expression of the entire HOXA cluster genes, exhibiting unique clinical and molecular features. For example, subgroup D is characterized by co-occurring NPM1 and TET2/IDH1/IDH2 mutations, older age, and high WBC/blast counts, while another HOX-subgroup (E) had monocytic nature and frequent RAS pathway mutations. By contrast, subgroups I and J were enriched for bi-allelic CEBPA mutations with and without frequent bZIP domain inframe mutations, respectively. GATA2 and WT1 mutations were common in subgroup I, while subgroup J was enriched for myelodysplasia-related mutations. TP53 mutations were largely clustered into subgroups (N, O and P) characterized by erythroid, immature progenitor, and tumor microenvironment cells, respectively. Additional ATAC subgroups included those having frequent RUNX1 (K), IDH1/IDH2 (L), and DDX41 (M) mutations, or showed a CMML-like AML phenotype (H). We next analyzed gene regulatory networks by combining RNA-seq and ATAC-seq, revealing key transcription factors (TFs) in each ATAC-subgroup. HOXA members played central roles in the HOX-related subgroups, while IRF members, including IRF4, 7, 8 and 9, were key TFs in the subgroup enriched for RUNX1 mutations (K), leading to the upregulation of the interferon pathway. ATAC subgroups also impacted patients' survival and significantly improved the risk prediction of ELN, enabling further stratification of each ELN risk group. Next, we performed an in vitro drug screening for 112 samples against 250 compounds and obtained a drug sensitivity profile for each ATAC-subgroup. As expected, the subgroup with frequent FLT3-ITD mutations showed a high sensitivity to a FLT3 inhibitor (quizartinib), while other subgroups (C, F, H) with monocytic differentiation and common RAS pathway mutations were sensitive to MEK inhibitors. Furthermore, we noted an unexpected sensitivity of subgroup K samples to multiple ABL inhibitors, even though they had no known ABL-related kinase mutations. To validate these findings, we predicted ATAC-subgroups for four external AML cohorts based on gene expression and successfully reproduced an equivalent ATAC-subgroups with similar clinical, genetic, and transcriptomic features as well as drug sensitivities. Finally, we performed single-cell RNA/ATAC-seq and profiled a total of 233,000 mononuclear cells from 31 patients. We observed that leukemic cells were separately clustered from normal cells, while cells from the same ATAC subgroups were co-clustered, supporting that leukemic cells had their own epigenetic profiles unique to each cluster. Conclusion Through a large-scale multi-omics analysis of AML, we revealed a comprehensive landscape of chromatin accessibility of AML, highlighting the role of epigenetic profiling as a powerful tool for deciphering heterogeneity of AML, which could be used for a better stratification of patients and therapeutics.

Consistent handling of samples is crucial for achieving reproducible molecular and functional testing results in translational research. Here, we used 229 acute myeloid leukemia (AML) patient samples to assess the impact of sample handling on high-throughput functional drug testing, mass spectrometry-based proteomics, and flow cytometry. Our data revealed novel and previously described changes in cell phenotype and drug response dependent on sample biobanking. Specifically, myeloid cells with a CD117 (c-KIT) positive phenotype decreased after biobanking, potentially distorting cell population representations and affecting drugs targeting these cells. Additionally, highly granular AML cell numbers decreased after freezing. Secondly, protein expression levels, as well as sensitivity to drugs targeting cell proliferation, metabolism, tyrosine kinases (e.g., JAK, KIT, FLT3), and BH3 mimetics were notably affected by biobanking. Moreover, drug response profiles of paired fresh and frozen samples showed that freezing samples can lead to systematic errors in drug sensitivity scores. While a high correlation between fresh and frozen for the entire drug library was observed, freezing cells had a considerable impact at an individual level, which could influence outcomes in translational studies. Our study highlights conditions where standardization is needed to improve reproducibility, and where validation of data generated from biobanked cohorts may be particularly important.

Acute myeloid leukemia (AML) is a disease caused by abnormal proliferations of myeloid progenitor cells in the bone marrow. About 25-30% of AML patients have a mutation in the FLT3 gene, which is associated with a poor prognosis. Although FLT3-inhibitors (FLT3i) that can target this mutation are clinically approved, approximately 40% of FLT3mut patients do not respond to FLT3i. In this study we performed high-throughput functional ex vivo drug testing (n=528) in 63 FLT3mut patients, with paired MS-proteomics (n=20), RNA-seq (n=20), plasma proteome (n=16), spatial single-cell proteomics (n=6) and mass cytometry (n=3) data with the aim to uncover the functional and molecular landscape linked to FLT3i response. First, we compared clinical and ex vivo response to FLT3i (midostaurin) to determine the clinical predictability of our ex vivo drug testing. All five ex vivo responders had a complete remission (CR) after first treatment with conventional induction therapy and midostaurin, while four out of five non-responders eventually relapsed or had no CR. Furthermore, non-responders had an increased immune activation and displayed immature stem cell phenotypes, as well as decreased expression of surface markers associated with mature myeloid cells (e.g., CD64, CD11c, CD33) compared to responders. Moreover, soluble CD40, CD244, PD-L1, CD4, and IL12RB1 were increased in responders, indicating an immune suppressive environment consistent with RNA and protein data. Surprisingly, the T cell receptor proteins CD200 and CD45RA were found to be increased in FLT3i responders and non-responders, respectively. None of the other 527 drugs tested ex vivo were more effective in the non-responder group, including other FLT3i. Thus, we performed drug combination screening on FLT3mut and FLT3i resistant cell lines and patient cells to identify effective drug combinations. Combinatorial drug screening in resistant FLT3mut cells revealed an increase in drug sensitivity to apoptotic modulators, as well as PI3K/AKT inhibitors in combination with midostaurin. Subsequent validation confirmed synergy between the midostaurin and the SMAC-mimetics birinapant and LCL161, while the BH3 mimetic venetoclax and the PI3K inhibitor idelalisib only showed an additive effect. Our data shows specific differences in myeloid maturation and LSC (Leukemic Stem Cell) phenotype between midostaurin responders and non-responders, together with a potential functional shift in cell signaling in immune signaling and anti-apoptotic pathways. Moreover, ex vivo drug testing data demonstrates that while there is less overall sensitivity to drug treatment in non-responders, combination therapies including apoptotic modulators such as the SMAC mimetics could overcome FLT3i resistance and improve FLT3mut patient outcomes.

Abstract The cancer hallmark concept defines biological properties that play a key role in cancer development and progression. The hallmarks reflect generic properties across all cancer types, and are not directly quantifiable nor applicable in cancer diagnostics. Here, we applied a data-driven approach to identify quantifiable hallmarks to be used as a basis for precision cancer medicine (PCM) in Acute Myeloid Leukemia (AML). We applied deep exome, transcriptome, DNA methylation and proteome profiling as well as ex-vivo functional testing of 525 drugs to 118 AML patient samples. Unsupervised multi-omic dimensionality reduction defined 11 independent axes of biological variability that we interpreted to reflect data-driven hallmarks (DDHM) of AML. Each DDHM integrates and ranks different data types and features, pinpointing those molecular features that were most informative for each hallmark. We calculated values for the 11 DDHM for each patient, constructing a DDHM-based precision medicine approach for AML diagnosis and therapy assignment. Most DDHMs were driven by other data types than genomics. For several DDHMs, different cytogenetic and mutational drivers converge on the same hallmarks and specific drug vulnerabilities. We also see how DDHM predictors of poor prognosis and high-risk AML are distinct from those that dictate specific drug response vulnerabilities. DDHM 2 reflected the cell differentiation path leading towards resistance to BH3 mimetics, ACK-inhibitors, and anthracyclines, and sensitivity to MEK inhibitors and TLR8 agonists. DDHM 2 derived a strong impact from protein expression of GATA2, MLLT11, DNMT3B and from RNA expression of WT1 and SOX4 as well as from multiple markers of different monocytic subtypes. DDHM 5 depicted cell cycle regulation and the Megakaryocytic-erythrocyte progenitor cell state that was enriched in AML patients with antecedent hematological disease. Interestingly, DDHM5 was linked to the sensitivity to purine analogs and vinca alkaloids. Moreover, DDHMs 1 and 8 captured clinical risk groups, prognosis, and responsiveness to hypomethylating agents. These DDHMs also revealed the phenotypic and functional biology that distinguished patients with high versus low FLT3-ITD mutant allele frequency. Validation of the DDHMs is realized through profiling and analysis of prospective AML samples, AML cell lines and previously published datasets. In summary, we present a data-driven approach for defining hallmarks in AML. The application of the DDHMs in AML provides a new paradigm for PCM and an opportunity for combinatorial therapeutic targeting. Each patient is characterized by a combination of independent and potentially druggable hallmarks, as opposed to traditional stratification in PCM, where each patient is assigned to one specific subgroup defined by genetic or other biomarkers. Citation Format: Tom Erkers, Nona Struyf, Tojo James, Francesco Marabita, Mattias Vesterlund, Nghia Vu, Cornelia Arnroth, Albin Österroos, Anna Bohlin, Sofia Bengtzén, Matthias Stahl, Rozbeh Jafari, Lukas Orre, Yudi Pawitan, Brinton Seashore-Ludlow, Janne Lehtiö, Sören Lehmann, Päivi Östling, Olli Kallioniemi. Data-driven hallmarks of cancer as a new paradigm for precision medicine: multi-omics and functional profiling in acute myeloid leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6612.

Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine (ara-C) and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that the addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-C triphosphate (ara-CTP) levels through targeted inhibition of SAMHD1. In this phase 1 trial, we evaluated the feasibility, safety and efficacy of the addition of hydroxyurea to standard chemotherapy with cytarabine/daunorubicin in newly diagnosed AML patients. Nine patients were enrolled and received at least two courses of ara-C (1 g/m 2 /2 h b.i.d. d1-5, i.e., a total of 10 g/m 2 per course), hydroxyurea (1–2 g d1-5) and daunorubicin (60 mg/m 2 d1-3). The primary endpoint was safety; secondary endpoints were complete remission rate and measurable residual disease (MRD). Additionally, pharmacokinetic studies of ara-CTP and ex vivo drug sensitivity assays were performed. The most common grade 3-4 toxicity was febrile neutropenia (100%). No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in median ara-CTP levels (1.5-fold; p = 0.04) in patients receiving doses of 1 g hydroxyurea. Ex vivo , diagnostic leukaemic bone marrow blasts from study patients were significantly sensitised to ara-C by a median factor of 2.1 ( p = 0.0047). All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow cytometry or real-time quantitative polymerase chain reaction [RT-qPCR]) had an MRD level <0.1% after two cycles of chemotherapy. Treatment was well-tolerated, and median time to neutrophil recovery >1.0 × 10 9 /L and to platelet recovery >50 × 10 9 /L after the start of cycle 1 was 19 days and 22 days, respectively. Six of nine patients underwent allogeneic haematopoietic stem-cell transplantation (allo-HSCT). With a median follow-up of 18.0 (range 14.9–20.5) months, one patient with adverse risk not fit for HSCT experienced a relapse after 11.9 months but is now in second complete remission. Targeted inhibition of SAMHD1 by the addition of hydroxyurea to conventional AML therapy is safe and appears efficacious within the limitations of the small phase 1 patient cohort. These results need to be corroborated in a larger study.

The cancer hallmark concept defines the biological processes that contribute to cancer development and progression. While the hallmarks represent an important theoretical framework, they reflect generic properties of all cancer types and hence most hallmarks are not measurable nor applicable to guiding diagnosis or therapy. Here, we carried out deep multi-omic profiling (DNA panel sequencing, , transcriptome, DNA methylation and proteome) and functional testing of responses to 525 drugs in 118 primary acute myeloid leukemia (AML). In addition, 47 primary AML patients were used as an independent validation cohort, including measurements by single-cell assays. Using unsupervised multi-omic dimensionality reduction, we defined 11 independent axes of biological variability that we considered here to reflect the data-driven hallmarks (DDHM) of AML. We defined the molecular features that contributed most to each hallmark, hence forming an understanding of the biological nature and pathways of each DDHM We hypothesized that the 11 DDHMs define biologically and clinically important features of AML applicable to diagnostics, therapy and precision medicine. For example, each patient had a unique composition of the DDHMs that reflected the AML biology of that patient case. Each DDHM was associated with distinct AML biology, including NPM1 mutation, transcriptional deregulation, stem cell properties, cell cycle, stromal signals, HOX signaling, and stress response. The DDHMs captured both biology that is already well known in AML, but with multi-omic resolution, and highlighted also novel, previously poorly understood, properties of AML. Each DDHM included a ranked list of drugs that explained the biology reflected in the hallmarks. For instance, the DDHM2 that defined sensitivity to Bcl-2 (Venetoclax) and MEK inhibitors highlighted myeloid cell differentiation patterns. Moreover, DDHM8 was strongly associated with patient prognosis, a higher response to hypomethylating agents and complete remission,. Combining this prognostic DDHM8 with hallmark DDHM1 that explained the omics of the NPM1 mutation landscape, captured the ELN risk classification and other prognostic cell surface markers. This revealed that several previously independently defined biomarkers of prognosis converge in the multi-omic paradigms defined by our DDHMs. In summary, we have defined through multi-omics and functional analysis a set of eleven data-driven hallmarks of AML. These capture some of the most important known prognostic and therapeutic biomarkers in AML but also highlight opportunities for novel biomarker discovery and drug repurposing. This approach provides a new quantitative and measurable framework to understand the biological heterogeneity of AML and exploit it for diagnosis, prognosis and precision therapy.

BACKGROUND:Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine (ara-C) and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that the addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-C triphosphate (ara-CTP) levels through targeted inhibition of SAMHD1. OBJECTIVES:In this phase 1 trial, we evaluated the feasibility, safety and efficacy of the addition of hydroxyurea to standard chemotherapy with cytarabine/daunorubicin in newly diagnosed AML patients. METHODS:Nine patients were enrolled and received at least two courses of ara-C (1 g/m2 /2 h b.i.d. d1-5, i.e., a total of 10 g/m2 per course), hydroxyurea (1-2 g d1-5) and daunorubicin (60 mg/m2 d1-3). The primary endpoint was safety; secondary endpoints were complete remission rate and measurable residual disease (MRD). Additionally, pharmacokinetic studies of ara-CTP and ex vivo drug sensitivity assays were performed. RESULTS:The most common grade 3-4 toxicity was febrile neutropenia (100%). No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in median ara-CTP levels (1.5-fold; p = 0.04) in patients receiving doses of 1 g hydroxyurea. Ex vivo, diagnostic leukaemic bone marrow blasts from study patients were significantly sensitised to ara-C by a median factor of 2.1 (p = 0.0047). All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow cytometry or real-time quantitative polymerase chain reaction [RT-qPCR]) had an MRD level <0.1% after two cycles of chemotherapy. Treatment was well-tolerated, and median time to neutrophil recovery >1.0 × 109 /L and to platelet recovery >50 × 109 /L after the start of cycle 1 was 19 days and 22 days, respectively. Six of nine patients underwent allogeneic haematopoietic stem-cell transplantation (allo-HSCT). With a median follow-up of 18.0 (range 14.9-20.5) months, one patient with adverse risk not fit for HSCT experienced a relapse after 11.9 months but is now in second complete remission. CONCLUSION:Targeted inhibition of SAMHD1 by the addition of hydroxyurea to conventional AML therapy is safe and appears efficacious within the limitations of the small phase 1 patient cohort. These results need to be corroborated in a larger study.

Functional testing on cancer patient samples is common in cancer research, but the methodology and type of samples used can vary considerably between studies. In 25 recent studies, where functional testing was performed on blood cancers, half used fresh samples, while others included only frozen or both sample types. Here, we investigated how sample handling influences functional testing in primary acute myeloid leukemia (AML) patient samples. To look for systematic changes in cell population induced by sample handling, we first compared cellular function between frozen (n=70) and freshly isolated (n=112) bone marrow mononuclear cell samples from patients with AML using a high-throughput functional drug testing assay with 528 clinically applied and emerging drugs. We then specifically collected paired samples (n=10) before and after cryopreservation using drug testing and flow cytometry as readouts, including a flow cytometry-based drug testing assay for selected compounds.The Z'-factor, an assay quality indicator, was similar between frozen and fresh sample groups. However, overall cell viability and growth rate were significantly lower in the frozen samples. Interestingly, in fresh samples, delays from sampling to the start of the assay had no effect up to 72h. When comparing overall drug efficacies, most of the drugs tested correlated highly between sample types, but 29 out of 528 drugs showed a notable systematic difference in drug sensitivity. Samples that had been frozen were more sensitive to drugs such as kinase inhibitors (amcasertib, dinaciclib), as well as apoptotic modulators (S-63845), and proteasome inhibitors (VLX1570). Additionally, fresh samples were more sensitive to paclitaxel than frozen ones across the paired samples.The cell composition of paired samples was also affected by cryopreservation, with a reduction in the frequency of cells expressing c-KIT (CD117) and a reduction of cells with high granularity (side scatter). Similar effects could be observed for the flow cytometry-based assay where frozen samples showed a cryopreservation-potentiated reduction in CD34+,CD11b+ and CD56+ cells after proteasome inhibitor treatment, and a reduction in CD56+ cells after taxane treatment. In conclusion, drug responses are in general highly correlated between fresh and frozen samples for about 95% of the drugs tested. However, careful consideration should be given to the specific drugs and cell populations of interest before deciding on sample handling methodology. This may be of particular importance when investigating specific drugs and phenotypes affecting cell proliferation and maturation stage, as sample handling may induce systematic differences and confound interpretation.

Background: Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-CTP levels through targeted inhibition of SAMHD1. Aims: To evaluate feasibility, safety, and efficacy of adding hydroxyurea to standard AML-directed therapy according to national guidelines. To perform translational studies including SAMHD1-staining on bone marrow sections, pharmacokinetics and drug-sensitivity analysis on leukemic cells ex vivo. Methods: This phase-1 trial (EudraCT-number: 2018-004050-16) was run at two sites (Karolinska University Hospital and Uppsala University Hospital, Sweden). Eligibility criteria included age >18 years, newly diagnosed non-promyelocytic AML, and fitness for intensive chemotherapy. Patients with CBF-AML eligible for treatment with gemtuzumab-ozogamicin were excluded. Treatment comprised 2 to 4 cycles of ara-C 1000 mg/m2 i.v. b.i.d. on day 1-5 during all 4 cycles and daunorubicin 60 mg/m2 i.v. q.d. on day 1-3 during cycles 1 and 2, and on day 1-2 during cycle 3. Patients with FLT3-mutated AML received midostaurin 50 mg b.i.d. on day 8-21 of each cycle. Risk-adapted allo-HSCT was performed at the discretion of the treating haematologist. The dose of hydroxyurea was escalated in a 3 + 3 design: 500 + 500 mg (level 1), 1000 + 500 mg (level 2), and 1000 + 1000 mg (level 3), each dose being given 1 hour prior to start of the ara-C infusion b.i.d. on day 1-5. Here we report the results of the first 9 patients in the run-in phase 1 part of the study. The phase 2 part will include an additional 60 patients. Recruitment is ongoing, utilizing the highest dose of hydroxyurea. Expression of SAMHD1 was assessed using a double-immunostaining method (SAMHD1/CD68), an autostainer system (BenchMark Ultra, Ventana, Rotkreuz, Switzerland) and previously validated protocols20. CD68+/SAMHD1+ histiocytes (macrophages) served as internal controls in all bone marrow biopsies assessed. Drug sensitivity analysis was performed on AML mononuclear cells utilized a high-throughput system evaluating >500 cytotoxic agents including combinations of ara-C and hydroxyurea in different doses. Results: All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow-cytometry or RT-qPCR) had an MRD level <0.1% after two cycles of chemotherapy. Six of nine patients underwent hematopoietic stem cell transplantation. With a median follow-up of 13.2 months, no relapse has been observed. No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in ara-CTP levels (1.5-fold; P=0.04) in the 6 patients receiving single doses of 1000 mg hydroxyurea. Drug-sensitivity analysis indicated an additive effect of ara-C and hydroxyurea on leukemic cells ex vivo. There was no apparent correlation between expression of SAMHD1-expression and efficacy; all patients had deep responses. Image:Summary/Conclusion: The high rate of complete remission and MRD negativity together with the pharmacokinetic and ex vivo evidence suggest that the efficacy of cytarabine-based AML treatment can be enhanced by addition of hydroxyurea as a targeted inhibitor of SAMHD1. Importantly, orally administered hydroxyurea may provide a safe, inexpensive, and broadly accessible strategy to improve outcome in AML. These results will have to be validated in a larger patient cohort.

Although copy number alterations (CNAs) and translocations constitute the backbone of the diagnosis and prognostication of acute myeloid leukemia (AML), techniques used for their assessment in routine diagnostics have not been reconsidered for decades. We used a combination of 2 next-generation sequencing-based techniques to challenge the currently recommended conventional cytogenetic analysis (CCA), comparing the approaches in a series of 281 intensively treated patients with AML. Shallow whole-genome sequencing (sWGS) outperformed CCA in detecting European Leukemia Net (ELN)-defining CNAs and showed that CCA overestimated monosomies and suboptimally reported karyotype complexity. Still, the concordance between CCA and sWGS for all ELN CNA-related criteria was 94%. Moreover, using in silico dilution, we showed that 1 million reads per patient would be enough to accurately assess ELN-defining CNAs. Total genomic loss, defined as a total loss ≥200 Mb by sWGS, was found to be a better marker for genetic complexity and poor prognosis compared with the CCA-based definition of complex karyotype. For fusion detection, the concordance between CCA and whole-transcriptome sequencing (WTS) was 99%. WTS had better sensitivity in identifying inv(16) and KMT2A rearrangements while showing limitations in detecting lowly expressed PML-RARA fusions. Ligation-dependent reverse transcription polymerase chain reaction was used for validation and was shown to be a fast and reliable method for fusion detection. We conclude that a next-generation sequencing-based approach can replace conventional CCA for karyotyping, provided that efforts are made to cover lowly expressed fusion transcripts.