Kmt2c mutations enhance HSC self-renewal capacity and convey a selective advantage after chemotherapy.

The myeloid tumor suppressor KMT2C is recurrently deleted in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), particularly therapy-related MDS/AML (t-MDS/t-AML), as part of larger chromosome 7 deletions. Here, we show that KMT2C deletions convey a selective advantage to hematopoietic stem cells (HSCs) after chemotherapy treatment that may precipitate t-MDS/t-AML. Kmt2c deletions markedly enhance murine HSC self-renewal capacity without altering proliferation rates. Haploid Kmt2c deletions convey a selective advantage only when HSCs are driven into cycle by a strong proliferative stimulus, such as chemotherapy. Cycling Kmt2c-deficient HSCs fail to differentiate appropriately, particularly in response to interleukin-1. Kmt2c deletions mitigate histone methylation/acetylation changes that accrue as HSCs cycle after chemotherapy, and they impair enhancer recruitment during HSC differentiation. These findings help explain why Kmt2c deletions are more common in t-MDS/t-AML than in de novo AML or clonal hematopoiesis: they selectively protect cycling HSCs from differentiation without inducing HSC proliferation themselves.

Single-Cell Analysis of Neonatal HSC Ontogeny Reveals Gradual and Uncoordinated Transcriptional Reprogramming that Begins before Birth.

Fetal and adult hematopoietic stem cells (HSCs) have distinct proliferation rates, lineage biases, gene expression profiles, and gene dependencies. Although these differences are widely recognized, it is not clear how the transition from fetal to adult identity is coordinated. Here we show that murine HSCs and committed hematopoietic progenitor cells (HPCs) undergo a gradual, rather than precipitous, transition from fetal to adult transcriptional states. The transition begins prior to birth and is punctuated by a late prenatal spike in type I interferon signaling that promotes perinatal HPC expansion and sensitizes progenitors to the leukemogenic FLT3ITD mutation. Most other changes in gene expression and enhancer activation are imprecisely timed and poorly coordinated. Thus, heterochronic enhancer elements, and their associated transcripts, are activated independently of one another rather than as part of a robust network. This simplifies the regulatory programs that guide neonatal HSC/HPC ontogeny, but it creates heterogeneity within these populations.

The efficiency of murine MLL-ENL-driven leukemia initiation changes with age and peaks during neonatal development.

MLL rearrangements are translocation mutations that cause both acute lymphoblastic leukemia and acute myeloid leukemia (AML). These translocations can occur as sole clonal driver mutations in infant leukemias, suggesting that fetal or neonatal hematopoietic progenitors may be exquisitely sensitive to transformation by MLL fusion proteins. To test this possibility, we used transgenic mice to induce one translocation product, MLL-ENL, during fetal, neonatal, juvenile and adult stages of life. When MLL-ENL was induced in fetal or neonatal mice, almost all died of AML. In contrast, when MLL-ENL was induced in adult mice, most survived for >1 year despite sustained transgene expression. AML initiation was most efficient when MLL-ENL was induced in neonates, and even transient suppression of MLL-ENL in neonates could prevent AML in most mice. MLL-ENL target genes were induced more efficiently in neonatal progenitors than in adult progenitors, consistent with the distinct AML initiation efficiencies. Interestingly, transplantation stress mitigated the developmental barrier to leukemogenesis. Since fetal/neonatal progenitors were highly competent to initiate MLL-ENL-driven AML, we tested whether Lin28b, a fetal master regulator, could accelerate leukemogenesis. Surprisingly, Lin28b suppressed AML initiation rather than accelerating it. This may explain why MLL rearrangements often occur before birth in human infant leukemia patients, but transformation usually does not occur until after birth, when Lin28b levels decline. Our findings show that the efficiency of MLL-ENL-driven AML initiation changes through the course of pre- and postnatal development, and developmental programs can be manipulated to impede transformation.

Exposure to maternal obesogenic diet worsens some but not all pre-cancer phenotypes in a murine genetic model of prostate cancer.

Prostate cancer research has been predominantly focused on adult exposures and risk factors. However, because the prostate develops during gestation and early life, exposure to external factors, such as obesity, during development could affect the prostate cancer progression in adults. Our previous work demonstrated that exposure to a high fat/high sugar (HF/HS) diet during gestation and until weaning stimulated prostate hyperplasia and altered the Pten/Akt pathway in adult mice fed a normal diet after weaning. Here, we asked whether maternal exposure to HF/HS would worsen prostate phenotypes in mice lacking Pten, a widely accepted driver of prostate cancer. We found that, at six weeks of age, both Chow (control)-and HF/HS-exposed Pten knockout mice showed evidence of murine PIN that included ducts with central comedo necrosis but that the HF/HS exposure did not influence murine PIN progression. The Pten knockout mice exposed to HF/HS in utero had significantly more mitotic cells than Pten knockouts exposed to Chow diet. In the Pten null background, the maternal HF/HS diet enhanced proliferation but did not have an additive effect on Akt activation. We observed neuroendocrine differentiation in Pten knockout mice, a phenotype that had not been previously described in this model.

Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing In Vivo.

Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in ∼ 11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in patients with MDS.

Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole-genome sequencing to perform an unbiased comprehensive screen to discover the somatic mutations in a sample from an individual with sAML and genotyped the loci containing these mutations in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (Ser34) in U2AF1 was recurrently present in 13 out of 150 (8.7%) subjects with de novo MDS, and we found suggestive evidence of an increased risk of progression to sAML associated with this mutation. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3' end of introns, and the alterations in U2AF1 are located in highly conserved zinc fingers of this protein. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This previously unidentified, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis.