A Weekly Low-Dose Regimen of Decitabine and Venetoclax is Efficacious and Less Myelotoxic in a Racially Diverse Cohort.

A metronomic, low-dose schedule of decitabine and Venetoclax was safe and effective in myeloid malignancies with few dose reductions or interruptions in an older diverse population. Median OS for AML and TP53 mutated patients was 16.1 and 11.3 months respectively.

Glutaminase inhibition in combination with azacytidine in myelodysplastic syndromes: a phase 1b/2 clinical trial and correlative analyses.

Malignancies are reliant on glutamine as an energy source and a facilitator of aberrant DNA methylation. We demonstrate preclinical synergy of telaglenastat (CB-839), a selective glutaminase inhibitor, combined with azacytidine (AZA), followed by a single-arm, open-label, phase 1b/2 study in persons with advanced myelodysplastic syndrome (MDS). The dual primary endpoints evaluated clinical activity, safety and tolerability; secondary endpoints evaluated pharmacokinetics, pharmacodynamics, overall survival, event-free survival and duration of response. The dose-escalation study included six participants and the dose-expansion study included 24 participants. Therapy was well tolerated and led to an objective response rate of 70% with (marrow) complete remission in 53% of participants and a median overall survival of 11.6 months, with evidence of myeloid differentiation in responders determined by single-cell RNA sequencing. Glutamine transporter solute carrier family 38 member 1 in MDS stem cells was associated with clinical responses and predictive of worse prognosis in a large MDS cohort. These data demonstrate the safety and efficacy of CB-839 and AZA as a combined metabolic and epigenetic approach in MDS. ClinicalTrials.gov identifier: NCT03047993 .

A STAT3 Degrader Demonstrates Pre-clinical Efficacy in Venetoclax resistant Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy that continues to have poor prognosis despite recent therapeutic advances. Venetoclax (Ven), a BCL2-inhibitor has shown a high response rate in AML; however, relapse is invariable due to mitochondrial dysregulation that includes upregulation of the antiapoptotic protein MCL1, a central mechanism of Ven resistance (Ven-res). We have previously demonstrated that the transcription factor STAT3 is upregulated in AML hematopoietic stem and progenitor cells (HSPCs) and can be effectively targeted to induce apoptosis of these aberrant cells. We now show that overexpression of STAT3 alone is sufficient to initiate a strong AML phenotype in a transgenic murine model. Phospho-proteomic data from Ven treated AML patients show a strong correlation of high total STAT3 and phospho-STAT3 [both p-STAT3(Y705) and p-STAT3(S727)] expression with worse survival and reduced remission duration. Additionally, significant upregulation of STAT3 was observed in Ven-res cell lines, in vivo models and primary patient samples. A novel and specific degrader of STAT3 demonstrated targeted reduction of total STAT3 and resulting inhibition of its active p-STAT3(Y705) and p-STAT3(S727) forms. Treatment with the STAT3 degrader induced apoptosis in parental and Ven-res AML cell lines and decreased mitochondrial depolarisation, and thereby dependency on MCL1 in Ven-res AML cell line, as observed by BH3 profiling assay. STAT3 degrader treatment also enhanced differentiation of myeloid and erythroid colonies in Ven-res peripheral blood mononuclear cells (PBMNCs). Upregulation of p-STAT3(S727) was also associated with pronounced mitochondrial structural and functional dysfunction in Ven-res cell lines, that were restored by STAT3 degradation. Treatment with a clinical-stage STAT3 degrader, KT-333 resulted in a significant reduction in STAT3 and MCL1 protein levels within two weeks of treatment in a cell derived xenograft model of Ven-res AML. Additionally, this treatment significant improvement in the survival of a Ven-res patient-derived xenograft in-vivo study. Degradation of STAT3 resulting in downregulation of MCL1 and improvements in global mitochondrial dysfunction suggests a novel mechanism of overcoming Ven-res in AML. Statement of Purpose: Five-year survival from AML is dismal at 30%. Our prior research demonstrated STAT3 over-expression in AML HSPC's to be associated with inferior survival. We now explore STAT3 over-expression in Ven-res AML, explain STAT3 mediated mitochondrial perturbations and describe a novel therapeutic strategy, STAT3 degradation to overcome Ven-res.

Splenomegaly and Response to Splenectomy in Immune Thrombocytopenia.

Background: Immune thrombocytopenia (ITP) is an acquired autoimmune disorder affecting patients of all ages and backgrounds. While current standards favor medical therapy in the frontline setting, splenectomy remains an integral part of treatment in refractory cases. Ideal parameters for patient selection for surgery remain elusive. Methods: Data for 40 adult patients undergoing splenectomy for ITP at a large urban center between 1 January 2010 and 1 July 2021 were collected and analyzed. Results: Most patients underwent uneventful laparoscopic splenectomy (95%). Complete or partial response at the time of last follow-up occurred in most patients (92.5%), with 60.0% requiring no additional medical therapy following surgery. Thrombosis was the predominant adverse event and the leading cause of death for two patients. Age and presence of splenomegaly appear to be associated with response to splenectomy. Conclusions: Splenectomy remains an effective therapy for selected patients with ITP. Predictors of positive response to splenectomy, such as younger age and the presence of splenomegaly, may help inform clinicians during patient selection for therapy. With strict attention paid to postoperative thromboprophylaxis, the diminishing use of splenectomy may not be warranted.

Socio-demographic determinants of myelofibrosis outcomes in an underserved center and the SEER national database.

The influence of demographic characteristics and social determinants on cancer outcomes is widely recognized in various malignancies but remains understudied in myelofibrosis (MF). This study aims to investigate social and demographic variables associated with MF survival. We retrospectively reviewed data of biopsy-proven MF patients from the Surveillance, Epidemiology and End Results (SEER) database (2000-2021) and Montefiore Medical Center (2000-2023), an underserved inner-city hospital. The SEER cohort included 5,403 MF patients and was predominantly Non-Hispanic (NH) White (82%) with a median age of 69 years. The age-adjusted incidence rate of MF was 0.32 cases per 100,000 person-years, increasing annually by 1.3% from 2000 to 2021. Two- and five- year overall survival rates were 69% and 42%, respectively. Worse cause-specific survival was associated with older age, male sex, and diagnosis before 2011 (year of Ruxolitinib approval). NH-Black ethnicity, unmarried status and lower median income were independent predictors of worse overall survival. The single-center analysis included 84 cases, with a median age of 66 years. NH-White patients comprised 37% of the sample, followed by NH-Black (28.5%). Two- and five- year overall survival rates were 90% and 61%, respectively, with NH-Black patients exhibiting the lowest median survival, although the difference was not statistically significant. Age was a significant predictor of worse survival in this cohort. NH-Black and Hispanic patients lived in areas with higher socioeconomic and demographic stress compared to NH-White patients. Overall, this study highlights the association of social and demographic factors with MF survival and emphasizes the need for equitable healthcare and further exploration of social-demographic factors affecting MF survival.

Immunotherapy-resistant acute lymphoblastic leukemia cells exhibit reduced CD19 and CD22 expression and BTK pathway dependency.

While therapies targeting CD19 by antibodies, chimeric antigen receptor T cells (CAR-T), and T cell engagers have improved the response rates in B cell malignancies, the emergence of resistant cell populations with low CD19 expression can lead to relapsed disease. We developed an in vitro model of adaptive resistance facilitated by chronic exposure of leukemia cells to a CD19 immunotoxin. Single-cell RNA-Seq (scRNA-Seq) showed an increase in transcriptionally distinct CD19lo populations among resistant cells. Mass cytometry demonstrated that CD22 was also decreased in these CD19lo-resistant cells. An assay for transposase-accessible chromatin with sequencing (ATAC-Seq) showed decreased chromatin accessibility at promoters of both CD19 and CD22 in the resistant cell populations. Combined loss of both CD19 and CD22 antigens was validated in samples from pediatric and young adult patients with B cell acute lymphoblastic leukemia (B-ALL) that relapsed after CD19 CAR-T-targeted therapy. Functionally, resistant cells were characterized by slower growth and lower basal levels of MEK activation. CD19lo resistant cells exhibited preserved B cell receptor signaling and were more sensitive to both Bruton's tyrosine kinase (BTK) and MEK inhibition. These data demonstrate that resistance to CD19 immunotherapies can result in decreased expression of both CD19 and CD22 and can result in dependency on BTK pathways.

Broad de-regulated U2AF1 splicing is prognostic and augments leukemic transformation via protein arginine methyltransferase activation.

The role of splicing dysregulation in cancer is underscored by splicing factor mutations; however, its impact in the absence of such rare mutations is poorly understood. To reveal complex patient subtypes and putative regulators of pathogenic splicing in Acute Myeloid Leukemia (AML), we developed a new approach called OncoSplice. Among diverse new subtypes, OncoSplice identified a biphasic poor prognosis signature that partially phenocopies U2AF1-mutant splicing, impacting thousands of genes in over 40% of adult and pediatric AML cases. U2AF1-like splicing co-opted a healthy circadian splicing program, was stable over time and induced a leukemia stem cell (LSC) program. Pharmacological inhibition of the implicated U2AF1-like splicing regulator, PRMT5, rescued leukemia mis-splicing and inhibited leukemic cell growth. Genetic deletion of IRAK4, a common target of U2AF1-like and PRMT5 treated cells, blocked leukemia development in xenograft models and induced differentiation. These analyses reveal a new prognostic alternative-splicing mechanism in malignancy, independent of splicing-factor mutations.

Impact of preexisting autoimmune disease on myelodysplastic syndromes outcomes: a population analysis.

Preexisting autoimmune disease affects between 10% and 30% of patients with myelodysplastic syndromes (MDS). Studies comparing outcomes in patients with MDS with and without autoimmune disease show discordant results. Using the Surveillance, Epidemiology, and End Results Medicare database, we conducted a population analysis to define the impact of autoimmunity on MDS outcomes. Cases were ascertained between 2007 and 2017 and claim algorithms used to identify autoimmune disease, demographic characteristics, comorbidity scores, MDS histology, transfusion burden, treatment with hypomethylating agents, and hematopoietic stem cell transplantation. Cox regression models estimated the impact on survival, and competing-risk regression models defined the effect on leukemic transformation. We analyzed 15 277 patients with MDS, including 2442 (16%) with preexisting autoimmune disease. The epidemiologic profile was distinctive in cases with preexisting autoimmunity, who were younger, were predominantly female, and had higher transfusion burden without difference in MDS histologic distribution. Autoimmune disease was associated with 11% decreased risk of death (hazard ratio [HR], 0.89; 95% confidence interval [CI], 0.85-0.94; P < .001). The effect on risk of leukemic transformation differed based on MDS histology. In low-risk MDS histologies, autoimmunity was associated with a 1.9-fold increased risk of leukemia (HR, 1.87; 95% CI, 1.17-2.99; P = .008), whereas no significant effect was seen in other groups. These results suggest that autoimmune disease affects survival in MDS and is associated with decreased mortality. The survival effect was evident in low-risk histologies despite higher risk of progression to leukemia. This could represent inflammation-driven hematopoiesis, simultaneously favoring less aggressive phenotypes and clonal expansion, which warrants further investigation.

Ribosomal protein control of hematopoietic stem cell transformation through direct, non-canonical regulation of metabolism.

We report here that expression of the ribosomal protein, RPL22, is frequently reduced in human myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML); reduced RPL22 expression is associated with worse outcomes. Mice null for Rpl22 display characteristics of an MDS-like syndrome and develop leukemia at an accelerated rate. Rpl22-deficient mice also display enhanced hematopoietic stem cell (HSC) self-renewal and obstructed differentiation potential, which arises not from reduced protein synthesis but from increased expression of the Rpl22 target, ALOX12, an upstream regulator of fatty acid oxidation (FAO). The increased FAO mediated by Rpl22-deficiency also persists in leukemia cells and promotes their survival. Altogether, these findings reveal that Rpl22 insufficiency enhances the leukemia potential of HSC via non-canonical de-repression of its target, ALOX12, which enhances FAO, a process that may serve as a therapeutic vulnerability of Rpl22 low MDS and AML leukemia cells. Highlights: RPL22 insufficiency is observed in MDS/AML and is associated with reduced survivalRpl22-deficiency produces an MDS-like syndrome and facilitates leukemogenesisRpl22-deficiency does not impair global protein synthesis by HSCRpl22 controls leukemia cell survival by non-canonical regulation of lipid oxidation eTOC: Rpl22 controls the function and transformation potential of hematopoietic stem cells through effects on ALOX12 expression, a regulator of fatty acid oxidation.

A Metabolically Optimized, Noncytotoxic Low-Dose Weekly Decitabine/Venetoclax in MDS and AML.

PURPOSE: Venetoclax (VEN) added to the hypomethylating agents (HMA) decitabine or azacitidine is the new standard of care for elderly patients with acute myeloid leukemia (AML) and is being evaluated in myelodysplastic syndrome (MDS). Current dosing of HMA/VEN relies on leukemia suppression through cytotoxicity which also impacts normal hematopoiesis. A regimen using once-weekly low-dose decitabine (LDDec) has demonstrated activity in myeloid malignancies. To overcome the severe myelosuppression often seen with HMA/VEN, we evaluated a once-weekly dosing regimen of VEN and LDDec in elderly and/or frail patients who were felt less likely to tolerate severe myelosuppression. PATIENTS AND METHODS: This is a retrospective, single-center analysis of patients with AML, MDS, or chronic myelomonocytic leukemia treated with a once-weekly LDDec/VEN regimen. We also compare this regimen with a cohort treated with standard dosing HMA/VEN. RESULTS: In a retrospective cohort of 39 patients, the overall response rate for patients receiving LDDec/VEN for first-line AML and MDS was 88% and 64%, respectively. In patients with TP53 mutations, the composite complete response rate was 71% and the median overall survival was 10.7 months. When compared with 36 patients receiving standard dose HMA/VEN, the LDDec/VEN patients had a longer time on therapy (175 vs. 78 days; P = 0.014) and a trend toward a higher rate of transfusion independence (47% vs. 26%; P = 0.33). Neutropenic fever occurred in 31% of patients, with a median of one hospitalization at any point during treatment. CONCLUSIONS: This preliminary clinical experience, although retrospective, provides proof-of-activity of noncytotoxic DNA methyltransferase 1-targeting by allowing frequent, sustained drug exposure often not possible with standard HMA/VEN regimens.

Initial development and testing of an exhaled microRNA detection strategy for lung cancer case-control discrimination.

For detecting field carcinogenesis non-invasively, early technical development and case-control testing of exhaled breath condensate microRNAs was performed. In design, human lung tissue microRNA-seq discovery was reconciled with TCGA and published tumor-discriminant microRNAs, yielding a panel of 24 upregulated microRNAs. The airway origin of exhaled microRNAs was topographically "fingerprinted", using paired EBC, upper and lower airway donor sample sets. A clinic-based case-control study (166 NSCLC cases, 185 controls) was interrogated with the microRNA panel by qualitative RT-PCR. Data were analyzed by logistic regression (LR), and by random-forest (RF) models. Feasibility testing of exhaled microRNA detection, including optimized whole EBC extraction, and RT and qualitative PCR method evaluation, was performed. For sensitivity in this low template setting, intercalating dye-based URT-PCR was superior to fluorescent probe-based PCR (TaqMan). In application, adjusted logistic regression models identified exhaled miR-21, 33b, 212 as overall case-control discriminant. RF analysis of combined clinical + microRNA models showed modest added discrimination capacity (1.1-2.5%) beyond clinical models alone: all subjects 1.1% (p = 8.7e-04)); former smokers 2.5% (p = 3.6e-05); early stage 1.2% (p = 9.0e-03), yielding combined ROC AUC ranging from 0.74 to 0.83. We conclude that exhaled microRNAs are qualitatively measureable, reflect in part lower airway signatures; and when further refined/quantitated, can potentially help to improve lung cancer risk assessment.

Study of efficacy and longevity of immune response to third and fourth doses of COVID-19 vaccines in patients with cancer: A single arm clinical trial.

Background: Cancer patients show increased morbidity with COVID-19 and need effective immunization strategies. Many healthcare regulatory agencies recommend administering 'booster' doses of COVID-19 vaccines beyond the standard two-dose series, for this group of patients. Therefore, studying the efficacy of these additional vaccine doses against SARS-CoV-2 and variants of concern is of utmost importance in this immunocompromised patient population. Methods: We conducted a prospective single arm clinical trial enrolling patients with cancer that had received two doses of mRNA or one dose of AD26.CoV2.S vaccine and administered a third dose of mRNA vaccine. We further enrolled patients that had no or low responses to three mRNA COVID vaccines and assessed the efficacy of a fourth dose of mRNA vaccine. Efficacy was assessed by changes in anti-spike antibody, T-cell activity, and neutralization activity, which were again assessed at baseline and 4 weeks. Results: We demonstrate that a third dose of COVID-19 vaccine leads to seroconversion in 57% of patients that were seronegative after primary vaccination series. The immune response is durable as assessed by anti-SARS-CoV-2 (anti-S) antibody titers, T-cell activity, and neutralization activity against wild-type (WT) SARS-CoV2 and BA1.1.529 at 6 months of follow-up. A subset of severely immunocompromised hematologic malignancy patients that were unable to mount an adequate immune response (titer <1000 AU/mL) after the third dose and were treated with a fourth dose in a prospective clinical trial which led to adequate immune boost in 67% of patients. Low baseline IgM levels and CD19 counts were associated with inadequate seroconversion. Booster doses induced limited neutralization activity against the Omicron variant. Conclusions: These results indicate that third dose of COVID vaccine induces durable immunity in cancer patients and an additional dose can further stimulate immunity in a subset of patients with inadequate response. Funding: Leukemia Lymphoma Society, National Cancer Institute. Clinical trial number: NCT05016622.

People with cancer have a higher risk of death or severe complications from COVID-19. As a result, vaccinating cancer patients against COVID-19 is critical. But patients with cancer, particularly blood or lymphatic system cancers, are less likely to develop protective immunity after COVID-19 vaccination. Immune suppression caused by cancer or cancer therapies may explain the poor vaccine response. Booster doses of the vaccine may improve the vaccine response in patients with cancer. But limited information is available about how well booster doses protect patients with cancer against COVID-19. Thakkar et al. show that a third dose of a COVID-19 vaccine can induce a protective immune response in half of the patients with cancer with no immunity after the first two doses. In the experiments, Thakkar et al. tracked the immune reaction to COVID-19 booster shots in 106 cancer patients. A third booster dose protected patients for up to four to six months and reduced breakthrough infection rates to low levels. Eighteen patients with blood cancers and severe immune suppression had an inadequate immune response after three doses of the vaccine; a fourth dose boosted the immune response for two-thirds of them, which for some included neutralization of variants such as Omicron. The experiments show that booster doses can increase COVID-19 vaccine protection for patients with cancer, even those who do not respond to the initial vaccine series. Thakkar et al. also show that pre-vaccine levels of two molecules linked to the immune system, (immunoglobin M and the CD19 antigen) predicted the patients' vaccine response, which might help physicians identify which individuals would benefit from booster doses.

Glutaminase inhibition in combination with azacytidine in myelodysplastic syndromes: Clinical efficacy and correlative analyses.

Malignancies can become reliant on glutamine as an alternative energy source and as a facilitator of aberrant DNA methylation, thus implicating glutaminase (GLS) as a potential therapeutic target. We demonstrate preclinical synergy of telaglenastat (CB-839), a selective GLS inhibitor, when combined with azacytidine (AZA), in vitro and in vivo, followed by a phase Ib/II study of the combination in patients with advanced MDS. Treatment with telaglenastat/AZA led to an ORR of 70% with CR/mCRs in 53% patients and a median overall survival of 11.6 months. scRNAseq and flow cytometry demonstrated a myeloid differentiation program at the stem cell level in clinical responders. Expression of non-canonical glutamine transporter, SLC38A1, was found to be overexpressed in MDS stem cells; was associated with clinical responses to telaglenastat/AZA and predictive of worse prognosis in a large MDS cohort. These data demonstrate the safety and efficacy of a combined metabolic and epigenetic approach in MDS.

PI3-kinase deletion promotes myelodysplasia by dysregulating autophagy in hematopoietic stem cells.

Myelodysplastic syndrome (MDS) is a clonal malignancy arising in hematopoietic stem cells (HSCs). The mechanisms of MDS initiation in HSCs are still poorly understood. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is frequently activated in acute myeloid leukemia, but in MDS, PI3K/AKT is often down-regulated. To determine whether PI3K down-regulation can perturb HSC function, we generated a triple knockout (TKO) mouse model with Pik3ca, Pik3cb, and Pik3cd deletion in hematopoietic cells. Unexpectedly, PI3K deficiency caused cytopenias, decreased survival, and multilineage dysplasia with chromosomal abnormalities, consistent with MDS initiation. TKO HSCs exhibit impaired autophagy, and pharmacologic autophagy induction improved HSC differentiation. Using intracellular LC3 and P62 flow cytometry and transmission electron microscopy, we also observed abnormal autophagic degradation in patient MDS HSCs. Therefore, we have uncovered an important protective role for PI3K in maintaining autophagic flux in HSCs to preserve the balance between self-renewal and differentiation and to prevent MDS initiation.

Lenalidomide and Eltrombopag for Treatment of Low- or Intermediate-Risk Myelodysplastic Syndrome: Result of a Phase II Clinical Trial.

PURPOSE: Thrombocytopenia is a serious complication of myelodysplastic syndromes (MDS) associated with an increased bleeding risk and worse prognosis. Eltrombopag (ELT), a thrombopoietin receptor agonist, can increase platelet counts and reverse anti-megakaryopoietic effects of lenalidomide (LEN) in preclinical studies. We hypothesized ELT would reduce the incidence of thrombocytopenia in MDS. PATIENTS AND METHODS: We conducted a Phase II multicenter trial of ELT and LEN in adult patients with low- or intermediate-1-risk MDS with symptomatic or transfusion-dependent anemia or thrombocytopenia (NCT01772420). Thrombocytopenic patients were started on ELT and subsequently treated with LEN after platelets were increased. Patients without thrombocytopenia were started on LEN monotherapy and treated with ELT if they became thrombocytopenic. RESULTS: Fifty-two patients were enrolled; mean age was 71 years (range 34-93). Overall response rate (ORR) in the intention-to-treat population was 35% (18/52). ELT monotherapy led to ORR of 33.3% (7/21), 29% achieving hematologic improvement (HI)-Platelets, and 24% bilineage responses. LEN monotherapy had 38% ORR (6/16) with all responders achieving HI-Erythroid. Fifteen patients received both ELT and LEN with ORR of 33.3%, 20% achieved HI-Erythroid, and 20% HI-Platelets with 13% bilineage responses. Median duration of response was 40 weeks for ELT (range 8-ongoing), 41 weeks (25-ongoing) for LEN, and 88 weeks (8.3-ongoing) for ELT/LEN. Non-hematologic grade 3-4 treatment-related adverse events were infrequent. Among patients on ELT, 2 had major bleeding events, 1 had a reversible increase in peripheral blasts, and 1 developed marrow fibrosis after 6 years on ELT. CONCLUSIONS: ELT and LEN are well tolerated and effective in achieving hematologic improvement in patients with low-/intermediate-risk MDS.

Splicing factor deficits render hematopoietic stem and progenitor cells sensitive to STAT3 inhibition.

Hematopoietic stem and progenitor cells (HSPCs) sustain lifelong hematopoiesis. Mutations of pre-mRNA splicing machinery, especially splicing factor 3b, subunit 1 (SF3B1), are early lesions found in malignancies arising from HSPC dysfunction. However, why splicing factor deficits contribute to HSPC defects remains incompletely understood. Using zebrafish, we show that HSPC formation in sf3b1 homozygous mutants is dependent on STAT3 activation. Clinically, mutations in SF3B1 are heterozygous; thus, we explored if targeting STAT3 could be a vulnerability in these cells. We show that SF3B1 heterozygosity confers heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Cells carrying mutations in other splicing factors or treated with splicing modulators are also more sensitive to STAT3 inhibition. Mechanistically, we illustrate that STAT3 inhibition exacerbates aberrant splicing in SF3B1 mutant cells. Our findings reveal a conserved vulnerability of splicing factor mutant HSPCs that could allow for their selective targeting in hematologic malignancies.

Activation of targetable inflammatory immune signaling is seen in myelodysplastic syndromes with SF3B1 mutations.

Background: Mutations in the SF3B1 splicing factor are commonly seen in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), yet the specific oncogenic pathways activated by mis-splicing have not been fully elucidated. Inflammatory immune pathways have been shown to play roles in the pathogenesis of MDS, though the exact mechanisms of their activation in splicing mutant cases are not well understood. Methods: RNA-seq data from SF3B1 mutant samples was analyzed and functional roles of interleukin-1 receptor-associated kinase 4 (IRAK4) isoforms were determined. Efficacy of IRAK4 inhibition was evaluated in preclinical models of MDS/AML. Results: RNA-seq splicing analysis of SF3B1 mutant MDS samples revealed retention of full-length exon 6 of IRAK4, a critical downstream mediator that links the Myddosome to inflammatory NF-kB activation. Exon 6 retention leads to a longer isoform, encoding a protein (IRAK4-long) that contains the entire death domain and kinase domain, leading to maximal activation of NF-kB. Cells with wild-type SF3B1 contain smaller IRAK4 isoforms that are targeted for proteasomal degradation. Expression of IRAK4-long in SF3B1 mutant cells induces TRAF6 activation leading to K63-linked ubiquitination of CDK2, associated with a block in hematopoietic differentiation. Inhibition of IRAK4 with CA-4948, leads to reduction in NF-kB activation, inflammatory cytokine production, enhanced myeloid differentiation in vitro and reduced leukemic growth in xenograft models. Conclusions: SF3B1 mutation leads to expression of a therapeutically targetable, longer, oncogenic IRAK4 isoform in AML/MDS models. Funding: This work was supported by Cincinnati Children's Hospital Research Foundation, Leukemia Lymphoma Society, and National Institute of Health (R35HL135787, RO1HL111103, RO1DK102759, RO1HL114582), Gabrielle's Angel Foundation for Cancer Research, and Edward P. Evans Foundation grants to DTS. AV is supported by Edward P. Evans Foundation, National Institute of Health (R01HL150832, R01HL139487, R01CA275007), Leukemia and Lymphoma Society, Curis and a gift from the Jane and Myles P. Dempsey family. AP and JB are supported by Blood Cancer UK (grants 13042 and 19004). GC is supported by a training grant from NYSTEM. We acknowledge support of this research from The Einstein Training Program in Stem Cell Research from the Empire State Stem Cell Fund through New York State Department of Health Contract C34874GG. MS is supported by a National Institute of Health Research Training and Career Development Grant (F31HL132420).

Genes contain blocks of code that tell cells how to make each part of a protein. Between these blocks are sections of linking DNA, which cells remove when they are preparing to use their genes. Scientists call this process 'splicing'. Cells can splice some genes in more than one way, allowing them to make different proteins from the same genetic code. Mutations that affect the splicing process can change the way cells make their proteins, leading to disease. For example, the myelodysplastic syndromes are a group of blood cancers often caused by mutations in splicing proteins, such as SF3B1. The disorder stops blood cells from maturing and causes abnormal inflammation. So far, the link between splicing, blood cell immaturity, inflammation and cancer is not clear. To find out more, Choudhary, Pellagatti et al. looked at the spliced genetic code from people with myelodysplastic syndromes. Mutations in the splicing protein SF3B1 changed the way cells spliced an important signalling molecule known as IRAK4. Affected cells cut out less genetic code and made a longer version of this signalling protein, named IRAK4-Long. This altered protein activated inflammation and stopped blood cells from maturing. Blocking IRAK4-Long reversed the effects. It also reduced tumour formation in mice carrying affected human cells. The molecule used to block IRAK4, CA-4948 ­ also known as Emavusertib ­ is currently being evaluated in clinical trials for myelodysplastic syndromes and other types of blood cancer. The work of Choudhary, Pellagatti et al. could help scientists to design genetic tests to predict which patients might benefit from this treatment.

PU.1-Dependent Enhancer Inhibition Separates Tet2-Deficient Hematopoiesis from Malignant Transformation.

Cytosine hypermethylation in and around DNA-binding sites of master transcription factors, including PU.1, occurs in aging hematopoietic stem cells following acquired loss-of-function mutations of DNA methyl-cytosine dioxygenase ten-eleven translocation-2 (TET2), albeit functional relevance has been unclear. We show that Tet2-deficient mouse hematopoietic stem and progenitor cells undergo malignant transformation upon compromised gene regulation through heterozygous deletion of an upstream regulatory region (UREΔ/WT) of the PU.1 gene. Although compatible with multilineage blood formation at young age, Tet2-deficient PU.1 UREΔ/WT mice develop highly penetrant, transplantable acute myeloid leukemia (AML) during aging. Leukemic stem and progenitor cells show hypermethylation at putative PU.1-binding sites, fail to activate myeloid enhancers, and are hallmarked by a signature of genes with impaired expression shared with human AML. Our study demonstrates that Tet2 and PU.1 jointly suppress leukemogenesis and uncovers a methylation-sensitive PU.1-dependent gene network as a unifying molecular vulnerability associated with AML. SIGNIFICANCE: We identify moderately impaired PU.1 mRNA expression as a biological modality predisposing Tet2-deficient hematopoietic stem and progenitor cells to malignant transformation. Our study furthermore uncovers a methylation-sensitive PU.1 gene network as a common feature of myeloid leukemia potentially allowing for the identification of patients at risk for malignant transformation. See related commentary by Schleicher and Pietras, p. 378. This article is highlighted in the In This Issue feature, p. 369.