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.

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.

Innate immune mediator, Interleukin-1 receptor accessory protein (IL1RAP), is expressed and pro-tumorigenic in pancreatic cancer.

Advanced pancreatic ductal adenocarcinoma (PDAC) is usually an incurable malignancy that needs newer therapeutic targets. Interleukin-1 receptor accessory protein (IL1RAP) is an innate immune mediator that regulates activation of pro-inflammatory and mitogenic signaling pathways. Immunohistochemistry on tissue microarrays demonstrated expression of IL1RAP in majority of human PDAC specimens and in murine pancreatic tumors from K-RasG122D/p53R172H/PDXCre (KPC) mice. Single cell RNA-Seq analysis of human primary pre-neoplastic lesions and adenocarcinoma specimens indicated that overexpression occurs during carcinogenesis. IL1RAP overexpression was associated with worse overall survival. IL1RAP knockdown significantly reduced cell viability, invasiveness, and clonogenic growth in pancreatic cancer cell lines. Inhibition of the downstream interleukin-1 receptor-associated kinase 4 (IRAK4) using two pharmacologic inhibitors, CA-4948 and PF06650833, resulted in reduced growth in pancreatic cancer cell lines and in xenograft models.

High burden of clonal hematopoiesis in first responders exposed to the World Trade Center disaster.

The terrorist attacks on the World Trade Center (WTC) created an unprecedented environmental exposure to aerosolized dust, gases and potential carcinogens. Clonal hematopoiesis (CH) is defined as the acquisition of somatic mutations in blood cells and is associated with smoking and exposure to genotoxic stimuli. Here we show that deep targeted sequencing of blood samples identified a significantly higher proportion of WTC-exposed first responders with CH (10%; 48 out of 481) when compared with non-WTC-exposed firefighters (6.7%; 17 out of 255; odds ratio, 3.14; 95% confidence interval, 1.64-6.03; P = 0.0006) after controlling for age, sex and race/ethnicity. The frequency of somatic mutations in WTC-exposed first responders showed an age-related increase and predominantly affected DNMT3A, TET2 and other CH-associated genes. Exposure of lymphoblastoid cells to WTC particulate matter led to dysregulation of DNA replication at common fragile sites in vitro. Moreover, mice treated with WTC particulate matter developed an increased burden of mutations in hematopoietic stem and progenitor cell compartments. In summary, the high burden of CH in WTC-exposed first responders provides a rationale for enhanced screening and preventative efforts in this population.

Efficacy of booster doses in augmenting waning immune responses to COVID-19 vaccine in patients with cancer.

Anti-COVID-19 immunity dynamics were assessed in patients with cancer in a prospective clinical trial. Waning of immunity was detected 4-6 months post-vaccination with significant increases in anti-spike IgG titers after booster dosing, and 56% of seronegative patients seroconverted post-booster vaccination. Prior anti-CD20/BTK inhibitor therapy was associated with reduced vaccine efficacy.

ASXL1 mutations are associated with distinct epigenomic alterations that lead to sensitivity to venetoclax and azacytidine.

The BCL2-inhibitor, Venetoclax (VEN), has shown significant anti-leukemic efficacy in combination with the DNMT-inhibitor, Azacytidine (AZA). To explore the mechanisms underlying the selective sensitivity of mutant leukemia cells to VEN and AZA, we used cell-based isogenic models containing a common leukemia-associated mutation in the epigenetic regulator ASXL1. KBM5 cells with CRISPR/Cas9-mediated correction of the ASXL1G710X mutation showed reduced leukemic growth, increased myeloid differentiation, and decreased HOXA and BCL2 gene expression in vitro compared to uncorrected KBM5 cells. Increased expression of the anti-apoptotic gene, BCL2, was also observed in bone marrow CD34+ cells from ASXL1 mutant MDS patients compared to CD34+ cells from wild-type MDS cases. ATAC-sequencing demonstrated open chromatin at the BCL2 promoter in the ASXL1 mutant KBM5 cells. BH3 profiling demonstrated increased dependence of mutant cells on BCL2. Upon treatment with VEN, mutant cells demonstrated increased growth inhibition. In addition, genome-wide methylome analysis of primary MDS samples and isogenic cell lines demonstrated increased gene-body methylation in ASXL1 mutant cells, with consequently increased sensitivity to AZA. These data mechanistically link the common leukemia-associated mutation ASXL1 to enhanced sensitivity to VEN and AZA via epigenetic upregulation of BCL2 expression and widespread alterations in DNA methylation.

First-in-human study of inhaled Azacitidine in patients with advanced non-small cell lung cancer.

BACKGROUND: Aerosolized Azacitidine has been shown to inhibit orthotopic lung cancer growth and induce re-expression of methylated tumor suppressor genes in murine models. We hypothesized that inhaled Azacitidine is safe and effective in reversing epigenetic changes in the bronchial epithelium secondary to chronic smoking. PATIENTS AND METHODS: We report the first in human study of inhaled Azacitidine. Azacitidine in aqueous solution was used to generate an aerosol suspension of 0.25-5 µm particle size. Main inclusion criteria: Stage IV or recurrent NSCLC with predominantly lung involvement, ≥1 prior systemic therapy, ECOG PS 0-1, and adequate pulmonary function. Patients received inhaled Azacitidine daily on days 1-5 and 15-19 of 28-day cycles, at 3 escalating doses (15, 30 and 45 mg/m2 daily). The primary objective was to determine the feasibility and tolerability of this new therapeutic modality. The key secondary objectives included pharmacokinetics, methylation profiles and efficacy. RESULTS: From 3/2015 to 2/2018, eight patients received a median number of 2 (IQR = 1) cycles of inhaled Azacitidine. No clinically significant adverse events were observed, except one patient treated at the highest dose developed an asymptomatic grade 2 decreased DLCO which resolved spontaneously. One patient receiving 12 cycles of therapy had an objective and durable partial response, and two patients had stable disease. Plasma Azacitidine was only briefly detectable in patients treated at the higher doses. Moreover, in 2 of 3 participants who agreed and underwent pre- and post-treatment bronchoscopy, the global DNA methylation in the bronchial epithelium decreased by 24 % and 79 % post-therapy, respectively. The interval between last inhaled treatment and bronchoscopy was 3 days. CONCLUSIONS: Inhaled Azacitidine resulted in negligible plasma levels compared to the previously reported subcutaneous administration and was well-tolerated. The results justify the continued development of inhaled Azacitidine at non-cytotoxic doses for patients with lung-confined malignant and/or premalignant lesions.

Biological basis for efficacy of activin receptor ligand traps in myelodysplastic syndromes.

Signaling by the TGF-ß superfamily is important in the regulation of hematopoiesis and is dysregulated in myelodysplastic syndromes (MDSs), contributing to ineffective hematopoiesis and clinical cytopenias. TGF-ß, activins, and growth differentiation factors exert inhibitory effects on red cell formation by activating canonical SMAD2/3 pathway signaling. In this Review, we summarize evidence that overactivation of SMAD2/3 signaling pathways in MDSs causes anemia due to impaired erythroid maturation. We also describe the basis for biological activity of activin receptor ligand traps, novel fusion proteins such as luspatercept that are promising as erythroid maturation agents to alleviate anemia and related comorbidities in MDSs and other conditions characterized by impaired erythroid maturation.

Stem cell mutations can be detected in myeloma patients years before onset of secondary leukemias.

Therapy-related acute myeloid leukemia and myelodysplastic syndromes (t-AML/t-MDS) are secondary hematologic malignancies associated with poor prognosis, warranting insights into their predisposing conditions and cells of origin. We identified patients with myeloma who developed t-AML/t-MDS and analyzed their stem and progenitor cells collected years before the onset of secondary disease. We demonstrate that aberrant stem cells with high CD123 expression can be detected long before the onset of overt leukemia. Rigorous sorting, followed by targeted sequencing, resulted in ultradeep functional depth of sequencing and revealed preexisting mutant hematopoietic stem cell (HSC) clones, mainly harboring TP53 mutations, that became the dominant population at the time of leukemic presentation. Taken together, these data show that HSCs can act as reservoirs for leukemia-initiating cells many years before the onset of myeloid leukemia.

Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts.

Even though pancreatic ductal adenocarcinoma (PDAC) is associated with fibrotic stroma, the molecular pathways regulating the formation of cancer associated fibroblasts (CAFs) are not well elucidated. An epigenomic analysis of patient-derived and de-novo generated CAFs demonstrated widespread loss of cytosine methylation that was associated with overexpression of various inflammatory transcripts including CXCR4. Co-culture of neoplastic cells with CAFs led to increased invasiveness that was abrogated by inhibition of CXCR4. Metabolite tracing revealed that lactate produced by neoplastic cells leads to increased production of alpha-ketoglutarate (aKG) within mesenchymal stem cells (MSCs). In turn, aKG mediated activation of the demethylase TET enzyme led to decreased cytosine methylation and increased hydroxymethylation during de novo differentiation of MSCs to CAF. Co-injection of neoplastic cells with TET-deficient MSCs inhibited tumor growth in vivo. Thus, in PDAC, a tumor-mediated lactate flux is associated with widespread epigenomic reprogramming that is seen during CAF formation.

Phase II Study of the ALK5 Inhibitor Galunisertib in Very Low-, Low-, and Intermediate-Risk Myelodysplastic Syndromes.

PURPOSE: Overactivation of TGF-ß signaling is observed in myelodysplastic syndromes (MDS) and is associated with dysplastic hematopoietic differentiation. Galunisertib, a first-in-class oral inhibitor of the TGF-ß receptor type 1 kinase (ALK5) has shown effectiveness in preclinical models of MDS and acceptable toxicity in phase I studies of solid malignancies. PATIENTS AND METHODS: A phase II multicenter study of galunisertib was conducted in patients with very low-, low-, or intermediate-risk MDS by the Revised International Prognostic Scoring System criteria with hemoglobin ≤ 10.0 g/dL. Patients received oral galunisertib 150 mg twice daily for 14 days on/14 days off. RESULTS: Ten of 41 evaluable patients (24.4%; 95% confidence interval, 12.4-40.3) achieved hematologic improvement erythroid response by International Working Group (IWG) 2006 criteria. A total of 18 of 41 patients (43.9%) achieved erythroid response as per IWG 2000 criteria. Nine of 28 (32.1%) of transfusion-dependent patients had hematologic improvement. A total of 18 of 41 (44%) patients had a significant reduction in fatigue. Overall median duration of response was 90 days in all patients. Rigorous stem and progenitor flow cytometry showed that patients with an early stem cell differentiation block were more likely to respond to galunisertib. The most common treatment-emergent adverse events were grade 1 or 2 in 20 (49%) of 41 patients, including any-grade fatigue (8/41, 20%), diarrhea (7/41, 17%), pyrexia (5/41, 12%), and vomiting (5/41, 12%). CONCLUSIONS: In summary, galunisertib treatment has an acceptable safety profile and was associated with hematologic improvements in lower- and intermediate-risk MDS, with responses in heavily transfusion-dependent patients and in those with signs of an early stem cell differentiation block.

Loss of Function of DOCK4 in Myelodysplastic Syndromes Stem Cells is Restored by Inhibitors of DOCK4 Signaling Networks.

PURPOSE: Myelodysplastic syndromes (MDS) with deletion of chromosome 7q/7 [-7/(del)7q MDS] is associated with worse outcomes and needs novel insights into pathogenesis. Reduced expression of signaling protein dedicator of cytokinesis 4 (DOCK4) in patients with -7/(del)7q MDS leads to a block in hematopoietic stem cell (HSC) differentiation. Identification of targetable signaling networks downstream of DOCK4 will provide means to restore hematopoietic differentiation in MDS.Experimental Design: We utilized phosphoproteomics approaches to identify signaling proteins perturbed as a result of reduced expression of DOCK4 in human HSCs and tested their functional significance in primary model systems. RESULTS: We demonstrate that reduced levels of DOCK4 lead to increased global tyrosine phosphorylation of proteins in primary human HSCs. LYN kinase and phosphatases INPP5D (SHIP1) and PTPN6 (SHP1) displayed greatest levels of tyrosine phosphorylation when DOCK4 expression levels were reduced using DOCK4-specific siRNA. Our data also found that increased phosphorylation of SHIP1 and SHP1 phosphatases were due to LYN kinase targeting these phosphatases as substrates. Increased migration and impediment of HSC differentiation were consequences of these signaling alterations. Pharmacologic inhibition of SHP1 reversed these functional aberrations in HSCs expressing low DOCK4 levels. In addition, differentiation block seen in DOCK4 haplo-insufficient [-7/(del)7q] MDS was rescued by inhibition of SHP1 phosphatase. CONCLUSIONS: LYN kinase and phosphatases SHP1 and SHIP1 are perturbed when DOCK4 expression levels are low. Inhibition of SHP1 promotes erythroid differentiation in healthy HSCs and in -7/(del)7q MDS samples with low DOCK4 expression. Inhibitors of LYN, SHP1 and SHIP1 also abrogated increased migratory properties in HSCs expressing reduced levels of DOCK4.

Misidentification of MLL3 and other mutations in cancer due to highly homologous genomic regions.

The MLL3 gene has been shown to be recurrently mutated in many malignancies including in families with acute myeloid leukemia. We demonstrate that many MLL3 variant calls made by exome sequencing are false positives due to misalignment to homologous regions, including a region on chr21, and can only be validated by long-range PCR. Numerous other recurrently mutated genes reported in COSMIC and TCGA databases have pseudogenes and cannot also be validated by conventional short read-based sequencing approaches. Genome-wide identification of pseudogene regions demonstrates that frequency of these homologous regions is increased with sequencing read lengths below 200 bps. To enable identification of poor quality sequencing variants in prospective studies, we generated novel genome-wide maps of regions with poor mappability that can be used in variant calling algorithms. Taken together, our findings reveal that pseudogene regions are a source of false-positive mutations in cancers.

U2AF1 mutations induce oncogenic IRAK4 isoforms and activate innate immune pathways in myeloid malignancies.

Spliceosome mutations are common in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but the oncogenic changes due to these mutations have not been identified. Here a global analysis of exon usage in AML samples revealed distinct molecular subsets containing alternative spliced isoforms of inflammatory and immune genes. Interleukin-1 receptor-associated kinase 4 (IRAK4) was the dominant alternatively spliced isoform in MDS and AML and is characterized by a longer isoform that retains exon 4, which encodes IRAK4-long (IRAK4-L), a protein that assembles with the myddosome, results in maximal activation of nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) and is essential for leukaemic cell function. Expression of IRAK4-L is mediated by mutant U2 small nuclear RNA auxiliary factor 1 (U2AF1) and is associated with oncogenic signalling in MDS and AML. Inhibition of IRAK4-L abrogates leukaemic growth, particularly in AML cells with higher expression of the IRAK4-L isoform. Collectively, mutations in U2AF1 induce expression of therapeutically targetable 'active' IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signalling in MDS and AML.

Cytokine-Regulated Phosphorylation and Activation of TET2 by JAK2 in Hematopoiesis.

Even though the Ten-eleven translocation (TET) enzymes catalyze the generation of 5-hydroxymethylcytosines required for lineage commitment and subsequent differentiation of stem cells into erythroid cells, the mechanisms that link extracellular signals to TET activation and DNA hydroxymethylation are unknown. We demonstrate that hematopoietic cytokines phosphorylate TET2, leading to its activation in erythroid progenitors. Specifically, cytokine receptor-associated JAK2 phosphorylates TET2 at tyrosines 1939 and 1964. Phosphorylated TET2 interacts with the erythroid transcription factor KLF1, and this interaction with TET2 is increased upon exposure to erythropoietin. The activating JAK2V617F mutation seen in myeloproliferative disease patient samples and in mouse models is associated with increased TET activity and cytosine hydroxymethylation as well as genome-wide loss of cytosine methylation. These epigenetic and functional changes are also associated with increased expression of several oncogenic transcripts. Thus, we demonstrate that JAK2-mediated TET2 phosphorylation provides a mechanistic link between extracellular signals and epigenetic changes during hematopoiesis. SIGNIFICANCE: Identification of TET2 phosphorylation and activation by cytokine-stimulated JAK2 links extracellular signals to chromatin remodeling during hematopoietic differentiation. This provides potential avenues to regulate TET2 function in the context of myeloproliferative disorders and myelodysplastic syndromes associated with the JAK2V617F-activating mutation.This article is highlighted in the In This Issue feature, p. 681.

Ascorbic acid-induced TET activation mitigates adverse hydroxymethylcytosine loss in renal cell carcinoma.

Although clear cell renal cell carcinoma (ccRCC) has been shown to result in widespread aberrant cytosine methylation and loss of 5-hydroxymethylcytosine (5hmC), the prognostic impact and therapeutic targeting of this epigenetic aberrancy has not been fully explored. Analysis of 576 primary ccRCC samples demonstrated that loss of 5hmC was strongly associated with aggressive clinicopathologic features and was an independent adverse prognostic factor. Loss of 5hmC also predicted reduced progression-free survival after resection of nonmetastatic disease. The loss of 5hmC in ccRCC was not due to mutational or transcriptional inactivation of ten eleven translocation (TET) enzymes, but to their functional inactivation by l-2-hydroxyglutarate (L2HG), which was overexpressed due to the deletion and underexpression of L2HG dehydrogenase (L2HGDH). Ascorbic acid (AA) reduced methylation and restored genome-wide 5hmC levels via TET activation. Fluorescence quenching of the recombinant TET-2 protein was unaffected by L2HG in the presence of AA. Pharmacologic AA treatment led to reduced growth of ccRCC in vitro and reduced tumor growth in vivo, with increased intratumoral 5hmC. These data demonstrate that reduced 5hmC is associated with reduced survival in ccRCC and provide a preclinical rationale for exploring the therapeutic potential of high-dose AA in ccRCC.

Antisense STAT3 inhibitor decreases viability of myelodysplastic and leukemic stem cells.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Transcriptomic analysis of stem and progenitor populations in MDS and AML demonstrated overexpression of STAT3 that was validated in an independent cohort. STAT3 overexpression was predictive of a shorter survival and worse clinical features in a large MDS cohort. High STAT3 expression signature in MDS CD34+ cells was similar to known preleukemic gene signatures. Functionally, STAT3 inhibition by a clinical, antisense oligonucleotide, AZD9150, led to reduced viability and increased apoptosis in leukemic cell lines. AZD9150 was rapidly incorporated by primary MDS/AML stem and progenitor cells and led to increased hematopoietic differentiation. STAT3 knockdown also impaired leukemic growth in vivo and led to decreased expression of MCL1 and other oncogenic genes in malignant cells. These studies demonstrate that STAT3 is an adverse prognostic factor in MDS/AML and provide a preclinical rationale for studies using AZD9150 in these diseases.

North American ATLL has a distinct mutational and transcriptional profile and responds to epigenetic therapies.

Adult T-cell leukemia lymphoma (ATLL) is a rare T cell neoplasm that is endemic in Japanese, Caribbean, and Latin American populations. Most North American ATLL patients are of Caribbean descent and are characterized by high rates of chemo-refractory disease and worse prognosis compared with Japanese ATLL. To determine genomic differences between these 2 cohorts, we performed targeted exon sequencing on 30 North American ATLL patients and compared the results with the Japanese ATLL cases. Although the frequency of TP53 mutations was comparable, the mutation frequency in epigenetic and histone modifying genes (57%) was significantly higher, whereas the mutation frequency in JAK/STAT and T-cell receptor/NF-κB pathway genes was significantly lower. The most common type of epigenetic mutation is that affecting EP300 (20%). As a category, epigenetic mutations were associated with adverse prognosis. Dissimilarities with the Japanese cases were also revealed by RNA sequencing analysis of 9 primary patient samples. ATLL samples with a mutated EP300 gene have decreased total and acetyl p53 protein and a transcriptional signature reminiscent of p53-mutated cancers. Most importantly, decitabine has highly selective single-agent activity in the EP300-mutated ATLL samples, suggesting that decitabine treatment induces a synthetic lethal phenotype in EP300-mutated ATLL cells. In conclusion, we demonstrate that North American ATLL has a distinct genomic landscape that is characterized by frequent epigenetic mutations that are targetable preclinically with DNA methyltransferase inhibitors.