Precision medicine in AML: overcoming resistance.

The development of molecularly targeted therapy for acute myeloid leukemia is progressing at an accelerated pace. Therapies targeting FLT3, IDH1, IDH2, and BCL2 have been approved in the last 5 years. As we exploit these biological vulnerabilities, various mechanisms of resistance arise. Emergence of competing clones with different genetic drivers and acquisition of constitutional mutations in the target renders therapies ineffective, and enzymatic isoform changes can lead to reappearance of the disease phenotype. Understanding the timing and circumstances of resistance origination will allow clinicians to develop combinatorial and sequential therapeutic approaches to deepen responses and improve survival. The objective of this review is to illustrate the biological underpinnings of each therapy and the landscape of resistance mechanisms and discuss strategies to overcome on- and off-target resistance.

Clonal evolution of hematopoietic stem cells after cancer chemotherapy.

Normal hematopoietic stem and progenitor cells (HSPCs) inherently accumulate somatic mutations and lose clonal diversity with age, processes implicated in the development of myeloid malignancies 1 . The impact of exogenous stressors, such as cancer chemotherapies, on the genomic integrity and clonal dynamics of normal HSPCs is not well defined. We conducted whole-genome sequencing on 1,032 single-cell-derived HSPC colonies from 10 patients with multiple myeloma (MM), who had undergone various chemotherapy regimens. Our findings reveal that melphalan treatment distinctly increases mutational burden with a unique mutation signature, whereas other MM chemotherapies do not significantly affect the normal mutation rate of HSPCs. Among these therapy-induced mutations were several oncogenic drivers such as TET2 and PPM1D . Phylogenetic analysis showed a clonal architecture in post-treatment HSPCs characterized by extensive convergent evolution of mutations in genes such as TP53 and PPM1D . Consequently, the clonal diversity and structure of post-treatment HSPCs mirror those observed in normal elderly individuals, suggesting an accelerated clonal aging due to chemotherapy. Furthermore, analysis of matched therapy-related myeloid neoplasm (t-MN) samples, which occurred 1-8 years later, enabled us to trace the clonal origin of t-MNs to a single HSPC clone among a group of clones with competing malignant potential, indicating the critical role of secondary mutations in dictating clonal dominance and malignant transformation. Our findings suggest that cancer chemotherapy promotes an oligoclonal architecture with multiple HSPC clones possessing competing leukemic potentials, setting the stage for the selective emergence of a singular clone that evolves into t-MNs after acquiring secondary mutations. These results underscore the importance of further systematic research to elucidate the long-term hematological consequences of cancer chemotherapy.

SOD1 is a synthetic-lethal target in PPM1D-mutant leukemia cells.

The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase Mg2+/Mn2+-dependent 1D) is a master negative regulator of the response; gain-of-function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacological target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D-mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D-mutant cells. Altogether, our results demonstrate a role for SOD1 in the survival of PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers.

Outcomes and genetic dynamics of acute myeloid leukemia at first relapse.

Patients with relapsed acute myeloid leukemia (rAML) experience dismal outcomes. We performed a comprehensive analysis of patients with rAML to determine the genetic dynamics and survival predictive factors. We analyzed 875 patients with newly diagnosed AML who received intensive treatment (IT) or low-intensity treatment (LIT). Of these patients, 197 experienced subsequent rAML. Data was available for 164 patients, with a median time from CR/CRi to relapse of 6.5 months. Thirty-five of the 164 patients (21%) experienced relapse after allogeneic hematopoietic stem cell transplantation (alloSCT). At relapse mutations in genes involved in pathway signaling tended to disappear, whereas clonal hematopoiesis-related mutations or TP53 tended to persist. Patients with normal karyotypes tended to acquire cytogenetic abnormalities at relapse. Patients treated with IT had a higher emergence rate of TP53 mutations (16%), compared to patients treated with LIT (1%, P = 0.009). The overall response rates were 38% and 35% for patients treated with salvage IT or LIT, respectively. Seventeen patients (10%) underwent alloSCT after salvage therapy. The median overall survival (OS) duration after relapse was 5.3 months, with a 1-year OS rate of 17.6%. Complex karyotype (hazard ratio [HR] = 2.14, P < 0.001), a KMT2A rearrangement (HR = 3.52, P = 0.011), time in remission < 12 months (HR = 1.71, P = 0.011), and an elevated white blood cell count at relapse (HR = 2.38, P = 0.005) were independent risk factors for OS duration. More effective frontline and maintenance therapies are warranted to prevent rAML.

Targeting MCL1-driven anti-apoptotic pathways overcomes blast progression after hypomethylating agent failure in chronic myelomonocytic leukemia.

RAS pathway mutations, which are present in 30% of patients with chronic myelomonocytic leukemia (CMML) at diagnosis, confer a high risk of resistance to and progression after hypomethylating agent (HMA) therapy, the current standard of care for the disease. Here, using single-cell, multi-omics technologies, we seek to dissect the biological mechanisms underlying the initiation and progression of RAS pathway-mutated CMML. We identify that RAS pathway mutations induce transcriptional reprogramming of hematopoietic stem and progenitor cells (HSPCs) and downstream monocytic populations in response to cell-intrinsic and -extrinsic inflammatory signaling that also impair the functions of immune cells. HSPCs expand at disease progression after therapy with HMA or the BCL2 inhibitor venetoclax and rely on the NF-κB pathway effector MCL1 to maintain survival. Our study has implications for the development of therapies to improve the survival of patients with RAS pathway-mutated CMML.

Therapy-related chronic myelomonocytic leukemia does not have the high-risk features of a therapy-related neoplasm.

ABSTRACT: Therapy-related myeloid neoplasms (t-MNs) arise after exposure to cytotoxic therapies and are associated with high-risk genetic features and poor outcomes. We analyzed a cohort of patients with therapy-related chronic myelomonocytic leukemia (tCMML; n = 71) and compared its features to that of de novo CMML (dnCMML; n = 461). Median time from cytotoxic therapy to tCMML diagnosis was 6.5 years. Compared with dnCMML, chromosome-7 abnormalities (4% vs 13%; P = .005) but not complex karyotype (3% vs 7%; P = .15), were more frequent in tCMML. tCMML was characterized by higher TP53 mutation frequency (4% vs 12%; P = .04) and lower NRAS (6% vs 22%, P = .007) and CBL (4% vs 12%, P = .04) mutation frequency. Prior therapy with antimetabolites (odd ratio [OR], 1.22; 95% confidence interval [CI], 1.05-1.42; P = .01) and mitotic inhibitors (OR, 1.24; 95% CI, 1.06-1.44; P = .009) was associated with NF1 and SETBP1 mutations whereas prior mitotic inhibitor therapy was associated with lower TET2 mutation frequency (OR, 0.71; 95% CI, 0.55-0.92; P = .01). Although no differences in median overall survival (OS) were observed among tCMML and dnCMML (34.7 months vs 35.9 months, P = .26), multivariate analysis for OS revealed that prior chemotherapy was associated with increased risk of death (hazard ratio, 1.76; 95% CI, 1.07-2.89; P = .026). Compared with a cohort of therapy-related myelodysplastic syndrome, tCMML had lower TP53 mutation frequency (12% vs 44.4%, P < .001) and less unfavorable outcomes. In summary, tCMML does not exhibit the high-risk features and poor outcomes of t-MNs.

Mutation order in acute myeloid leukemia identifies uncommon patterns of evolution and illuminates phenotypic heterogeneity.

Acute myeloid leukemia (AML) has a poor prognosis and a heterogeneous mutation landscape. Although common mutations are well-studied, little research has characterized how the sequence of mutations relates to clinical features. Using published, single-cell DNA sequencing data from three institutions, we compared clonal evolution patterns in AML to patient characteristics, disease phenotype, and outcomes. Mutation trees, which represent the order of select mutations, were created for 207 patients from targeted panel sequencing data using 1 639 162 cells, 823 mutations, and 275 samples. In 224 distinct orderings of mutated genes, mutations related to DNA methylation typically preceded those related to cell signaling, but signaling-first cases did occur, and had higher peripheral cell counts, increased signaling mutation homozygosity, and younger patient age. Serial sample analysis suggested that NPM1 and DNA methylation mutations provide an advantage to signaling mutations in AML. Interestingly, WT1 mutation evolution shared features with signaling mutations, such as WT1-early being proliferative and occurring in younger individuals, trends that remained in multivariable regression. Some mutation orderings had a worse prognosis, but this was mediated by unfavorable mutations, not mutation order. These findings add a dimension to the mutation landscape of AML, identifying uncommon patterns of leukemogenesis and shedding light on heterogeneous phenotypes.

BRG1/BRM inhibitor targets AML stem cells and exerts superior preclinical efficacy combined with BET or menin inhibitor.

ABSTRACT: BRG1 (SMARCA4) and BRM (SMARCA2) are the mutually exclusive core ATPases of the chromatin remodeling BAF (BRG1/BRM-associated factor) complexes. They enable transcription factors/cofactors to access enhancers/promoter and modulate gene expressions responsible for cell growth and differentiation of acute myeloid leukemia (AML) stem/progenitor cells. In AML with MLL1 rearrangement (MLL1r) or mutant NPM1 (mtNPM1), although menin inhibitor (MI) treatment induces clinical remissions, most patients either fail to respond or relapse, some harboring menin mutations. FHD-286 is an orally bioavailable, selective inhibitor of BRG1/BRM under clinical development in AML. Present studies show that FHD-286 induces differentiation and lethality in AML cells with MLL1r or mtNPM1, concomitantly causing perturbed chromatin accessibility and repression of c-Myc, PU.1, and CDK4/6. Cotreatment with FHD-286 and decitabine, BET inhibitor (BETi) or MI, or venetoclax synergistically induced in vitro lethality in AML cells with MLL1r or mtNPM1. In models of xenografts derived from patients with AML with MLL1r or mtNPM1, FHD-286 treatment reduced AML burden, improved survival, and attenuated AML-initiating potential of stem-progenitor cells. Compared with each drug, cotreatment with FHD-286 and BETi, MI, decitabine, or venetoclax significantly reduced AML burden and improved survival, without inducing significant toxicity. These findings highlight the FHD-286-based combinations as a promising therapy for AML with MLL1r or mtNPM1.

Cancer patients with clonal hematopoiesis die from primary malignancy or comorbidities despite higher rates of transformation to myeloid neoplasms.

BACKGROUND: The occurrence of somatic mutations in patients with no evidence of hematological disorders is called clonal hematopoiesis (CH). CH, whose subtypes include CH of indeterminate potential and clonal cytopenia of undetermined significance, has been associated with both hematologic cancers and systemic comorbidities. However, CH's effect on patients, especially those with concomitant malignancies, is not fully understood. METHODS: We performed a retrospective evaluation of all patients with CH at a tertiary cancer center. Patient characteristics, mutational data, and outcomes were collected and analyzed. RESULTS: Of 78 individuals included, 59 (76%) had a history of cancer and 60 (77%) had moderate to severe comorbidity burdens. DNMT3A, TET2, TP53, and ASXL1 were the most common mutations. For the entire cohort, the 2-year overall survival rate was 79% (95% CI: 70, 90), while the median survival was not reached. Of 20 observed deaths, most were related to primary malignancies (n = 7, 35%), comorbidities (n = 4, 20%), or myeloid neoplasms (n = 4, 20%). Twelve patients (15%) experienced transformation to a myeloid neoplasm. According to the clonal hematopoiesis risk score, the 3-year transformation rate was 0% in low-risk, 15% in intermediate-risk (p = 0.098), and 28% in high-risk (p = 0.05) patients. By multivariate analysis, transformation was associated with variant allele frequency ≥0.2 and hemoglobin <10 g/dL. CONCLUSIONS: In a population including mostly cancer patients, CH was associated with comorbidities and myeloid transformation in patients with higher mutational burdens and anemia. Nevertheless, such patients were less likely to die of their myeloid neoplasm than of primary malignancy or comorbidities.

Transomics2cytoscape: an automated software for interpretable 2.5-dimensional visualization of trans-omic networks.

Biochemical network visualization is one of the essential technologies for mechanistic interpretation of omics data. In particular, recent advances in multi-omics measurement and analysis require the development of visualization methods that encompass multiple omics data. Visualization in 2.5 dimension (2.5D visualization), which is an isometric view of stacked X-Y planes, is a convenient way to interpret multi-omics/trans-omics data in the context of the conventional layouts of biochemical networks drawn on each of the stacked omics layers. However, 2.5D visualization of trans-omics networks is a state-of-the-art method that primarily relies on time-consuming human efforts involving manual drawing. Here, we present an R Bioconductor package 'transomics2cytoscape' for automated visualization of 2.5D trans-omics networks. We confirmed that transomics2cytoscape could be used for rapid visualization of trans-omics networks presented in published papers within a few minutes. Transomics2cytoscape allows for frequent update/redrawing of trans-omics networks in line with the progress in multi-omics/trans-omics data analysis, thereby enabling network-based interpretation of multi-omics data at each research step. The transomics2cytoscape source code is available at https://github.com/ecell/transomics2cytoscape .

Efficacy of novel agents against cellular models of familial platelet disorder with myeloid malignancy (FPD-MM).

Germline, mono-allelic mutations in RUNX1 cause familial platelet disorder (RUNX1-FPD) that evolves into myeloid malignancy (FPD-MM): MDS or AML. FPD-MM commonly harbors co-mutations in the second RUNX1 allele and/or other epigenetic regulators. Here we utilized patient-derived (PD) FPD-MM cells and established the first FPD-MM AML cell line (GMR-AML1). GMR-AML1 cells exhibited active super-enhancers of MYB, MYC, BCL2 and CDK6, augmented expressions of c-Myc, c-Myb, EVI1 and PLK1 and surface markers of AML stem cells. In longitudinally studied bone marrow cells from a patient at FPD-MM vs RUNX1-FPD state, we confirmed increased chromatin accessibility and mRNA expressions of MYB, MECOM and BCL2 in FPD-MM cells. GMR-AML1 and PD FPD-MM cells were sensitive to homoharringtonine (HHT or omacetaxine) or mebendazole-induced lethality, associated with repression of c-Myc, EVI1, PLK1, CDK6 and MCL1. Co-treatment with MB and the PLK1 inhibitor volasertib exerted synergistic in vitro lethality in GMR-AML1 cells. In luciferase-expressing GMR-AML1 xenograft model, MB, omacetaxine or volasertib monotherapy, or co-treatment with MB and volasertib, significantly reduced AML burden and improved survival in the immune-depleted mice. These findings highlight the molecular features of FPD-MM progression and demonstrate HHT, MB and/or volasertib as effective agents against cellular models of FPD-MM.

Ivosidenib significantly reduces triazole levels in patients with acute myeloid leukemia and myelodysplastic syndrome.

BACKGROUND: Ivosidenib is primarily metabolized by CYP3A4; however, it induces CYP450 isozymes, including CYP3A4 and CYP2C9, whereas it inhibits drug transporters, including P-glycoprotein. Patients with acute myeloid leukemia are at risk of invasive fungal infections, and therefore posaconazole and voriconazole are commonly used in this population. Voriconazole is a substrate of CYP2C9, CYP2C19, and CYP3A4; therefore, concomitant ivosidenib may result in decreased serum concentrations. Although posaconazole is a substrate of P-glycoprotein, it is metabolized primarily via UDP glucuronidation; thus, the impact of ivosidenib on posaconazole exposure is unknown. METHODS: Patients treated with ivosidenib and concomitant triazole with at least one serum trough level were included. Subtherapeutic levels were defined as posaconazole <700 ng/mL and voriconazole <1.0 µg/mL. The incidences of breakthrough invasive fungal infections and QTc prolongation were identified at least 5 days after initiation of ivosidenib with concomitant triazole. RESULTS: Seventy-eight serum triazole levels from 31 patients receiving ivosidenib-containing therapy and concomitant triazole were evaluated. Of the 78 concomitant levels, 47 (60%) were subtherapeutic (posaconazole: n = 20 of 43 [47%]; voriconazole: n = 27 of 35 [77%]). Compared to levels drawn while patients were off ivosidenib, median triazole serum levels during concomitant ivosidenib were significantly reduced. There was no apparent increase in incidence of grade 3 QTc prolongation with concomitant azole antifungal and ivosidenib 500 mg daily. CONCLUSIONS: This study demonstrated that concomitant ivosidenib significantly reduced posaconazole and voriconazole levels. Voriconazole should be avoided, empiric high-dose posaconazole (>300 mg/day) may be considered, and therapeutic drug monitoring is recommended in all patients receiving concomitant ivosidenib.

Targeting DNA2 overcomes metabolic reprogramming in multiple myeloma.

DNA damage resistance is a major barrier to effective DNA-damaging therapy in multiple myeloma (MM). To discover mechanisms through which MM cells overcome DNA damage, we investigate how MM cells become resistant to antisense oligonucleotide (ASO) therapy targeting Interleukin enhancer binding factor 2 (ILF2), a DNA damage regulator that is overexpressed in 70% of MM patients whose disease has progressed after standard therapies have failed. Here, we show that MM cells undergo adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage activation. Using a CRISPR/Cas9 screening strategy, we identify the mitochondrial DNA repair protein DNA2, whose loss of function suppresses MM cells' ability to overcome ILF2 ASO-induced DNA damage, as being essential to counteracting oxidative DNA damage. Our study reveals a mechanism of vulnerability of MM cells that have an increased demand for mitochondrial metabolism upon DNA damage activation.

Increased iron uptake by splenic hematopoietic stem cells promotes TET2-dependent erythroid regeneration.

Hematopoietic stem cells (HSCs) are capable of regenerating the blood system, but the instructive cues that direct HSCs to regenerate particular lineages lost to the injury remain elusive. Here, we show that iron is increasingly taken up by HSCs during anemia and induces erythroid gene expression and regeneration in a Tet2-dependent manner. Lineage tracing of HSCs reveals that HSCs respond to hemolytic anemia by increasing erythroid output. The number of HSCs in the spleen, but not bone marrow, increases upon anemia and these HSCs exhibit enhanced proliferation, erythroid differentiation, iron uptake, and TET2 protein expression. Increased iron in HSCs promotes DNA demethylation and expression of erythroid genes. Suppressing iron uptake or TET2 expression impairs erythroid genes expression and erythroid differentiation of HSCs; iron supplementation, however, augments these processes. These results establish that the physiological level of iron taken up by HSCs has an instructive role in promoting erythroid-biased differentiation of HSCs.

Mapping Injection Order Messages to Health Level 7 Fast Healthcare Interoperability Resources to Collate Infusion Pump Data.

BACKGROUND: When administering an infusion to a patient, it is necessary to verify that the infusion pump settings are in accordance with the injection orders provided by the physician. However, the infusion rate entered into the infusion pump by the health care provider cannot be automatically reconciled with the injection order information entered into the electronic medical records (EMRs). This is because of the difficulty in linking the infusion rate entered into the infusion pump by the health care provider with the injection order information entered into the EMRs. OBJECTIVES: This study investigated a data linkage method for reconciling infusion pump settings with injection orders in the EMRs. METHODS: We devised and implemented a mechanism to convert injection order information into the Health Level 7 Fast Healthcare Interoperability Resources (FHIR), a new health information exchange standard, and match it with an infusion pump management system in a standard and simple manner using a REpresentational State Transfer (REST) application programming interface (API). The injection order information was extracted from Standardized Structured Medical Record Information Exchange version 2 International Organization for Standardization/technical specification 24289:2021 and was converted to the FHIR format using a commercially supplied FHIR conversion module and our own mapping definition. Data were also sent to the infusion pump management system using the REST Web API. RESULTS: Information necessary for injection implementation in hospital wards can be transferred to FHIR and linked. The infusion pump management system application screen allowed the confirmation that the two pieces of information matched, and it displayed an error message if they did not. CONCLUSION: Using FHIR, the data linkage between EMRs and infusion pump management systems can be smoothly implemented. We plan to develop a new mechanism that contributes to medical safety through the actual implementation and verification of this matching system.

Seasonal and diurnal variations in soil respiration rates at a treeline ecotone and a lower distribution limit of subalpine forests.

This study examined the seasonal and diurnal variations in soil respiration rates (RS) during a growing season at the treeline ecotone (2,800 m) and the lower distribution limit (1,600 m) of subalpine forests on a volcanic mountain in Japan. The aboveground biomass, the total RS during the growing season, and the RS per day during the growing season were lower at 2,800 m than those at 1,600 m. Seasonal RS variations positively correlated with those of soil and air temperatures at both elevations, and this tendency was more apparent at 1,600 m than 2,800 m. The mean volumetric soil water content (WS) during the growing season was much lower at 2,800 m than 1,600 m because of the scoria substrate at 2,800 m. The monthly mean diel cycle of RS was positively correlated with the soil temperature at each elevation every month, whereas that at 1,600 m was negatively correlated with that of the WS. The RS at 2,800 m decreased during the daytime especially in August, despite no changes in the WS. The decrease in RS after precipitation at 1,600 m was higher than that at 2,800 m. Seasonal and diurnal RS variations could be reproduced from soil and air temperatures, and WS. Estimating soil respiration rate from these variables will help understand the future carbon budget of forests due to global warming.