PPM1D modulates hematopoietic cell fitness and response to DNA damage and is a therapeutic target in myeloid malignancy.

PPM1D encodes a phosphatase that is recurrently activated across cancer, most notably in therapy-related myeloid neoplasms. However, the function of PPM1D in hematopoiesis and its contribution to tumor cell growth remain incompletely understood. Using conditional mouse models, we uncover a central role for Ppm1d in hematopoiesis and validate its potential as a therapeutic target. We find that Ppm1d regulates the competitive fitness and self-renewal of hematopoietic stem cells (HSCs) with and without exogenous genotoxic stresses. We also show that although Ppm1d activation confers cellular resistance to cytotoxic therapy, it does so to a lesser degree than p53 loss, informing the clonal competition phenotypes often observed in human studies. Notably, loss of Ppm1d sensitizes leukemias to cytotoxic therapies in vitro and in vivo, even in the absence of a Ppm1d mutation. Vulnerability to PPM1D inhibition is observed across many cancer types and dependent on p53 activity. Importantly, organism-wide loss of Ppm1d in adult mice is well tolerated, supporting the tolerability of pharmacologically targeting PPM1D. Our data link PPM1D gain-of-function mutations to the clonal expansion of HSCs, inform human genetic observations, and support the therapeutic targeting of PPM1D in cancer.

Clonal haematopoiesis and risk of chronic liver disease.

Chronic liver disease is a major public health burden worldwide1. Although different aetiologies and mechanisms of liver injury exist, progression of chronic liver disease follows a common pathway of liver inflammation, injury and fibrosis2. Here we examined the association between clonal haematopoiesis of indeterminate potential (CHIP) and chronic liver disease in 214,563 individuals from 4 independent cohorts with whole-exome sequencing data (Framingham Heart Study, Atherosclerosis Risk in Communities Study, UK Biobank and Mass General Brigham Biobank). CHIP was associated with an increased risk of prevalent and incident chronic liver disease (odds ratio = 2.01, 95% confidence interval (95% CI) [1.46, 2.79]; P < 0.001). Individuals with CHIP were more likely to demonstrate liver inflammation and fibrosis detectable by magnetic resonance imaging compared to those without CHIP (odds ratio = 1.74, 95% CI [1.16, 2.60]; P = 0.007). To assess potential causality, Mendelian randomization analyses showed that genetic predisposition to CHIP was associated with a greater risk of chronic liver disease (odds ratio = 2.37, 95% CI [1.57, 3.6]; P < 0.001). In a dietary model of non-alcoholic steatohepatitis, mice transplanted with Tet2-deficient haematopoietic cells demonstrated more severe liver inflammation and fibrosis. These effects were mediated by the NLRP3 inflammasome and increased levels of expression of downstream inflammatory cytokines in Tet2-deficient macrophages. In summary, clonal haematopoiesis is associated with an elevated risk of liver inflammation and chronic liver disease progression through an aberrant inflammatory response.

Degradation of GSPT1 causes TP53-independent cell death in leukemia while sparing normal hematopoietic stem cells.

Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early-phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR/Cas9 screens implicated decreased translation initiation as protective following GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to the effects of GSPT1 degradation. We defined 2 Crbn amino acids that prevent Gspt1 degradation in mice, generated a knockin mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant acute myeloid leukemia.

Lenalidomide promotes the development of TP53-mutated therapy-related myeloid neoplasms.

There is a growing body of evidence that therapy-related myeloid neoplasms (t-MNs) with driver gene mutations arise in the background of clonal hematopoiesis (CH) under the positive selective pressure of chemo- and radiation therapies. Uncovering the exposure relationships that provide selective advantage to specific CH mutations is critical to understanding the pathogenesis and etiology of t-MNs. In a systematic analysis of 416 patients with t-MN and detailed prior exposure history, we found that TP53 mutations were significantly associated with prior treatment with thalidomide analogs, specifically lenalidomide. We demonstrated experimentally that lenalidomide treatment provides a selective advantage to Trp53-mutant hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, the effect of which was specific to Trp53-mutant HSPCs and was not observed in HSPCs with other CH mutations. Because of the differences in CK1α degradation, pomalidomide treatment did not provide an equivalent level of selective advantage to Trp53-mutant HSPCs, providing a biological rationale for its use in patients at high risk for t-MN. These findings highlight the role of lenalidomide treatment in promoting TP53-mutated t-MNs and offer a potential alternative strategy to mitigate the risk of t-MN development.

Genetic barcoding systematically compares genes in del(5q) MDS and reveals a central role for CSNK1A1 in clonal expansion.

How genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSCs) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, we performed a systematic comparison on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single-clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1-/+ multipotent progenitors showed a proproliferative gene signature and HSCs showed a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1-/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. We therefore propose a crucial role for Csnk1a1 haploinsufficiency in the selective advantage of 5q-HSCs, implemented by creation of a unique competitive advantage through increased HSC self-renewal and proliferation capacity, as well as increased fitness under inflammatory stress.

Association of clonal hematopoiesis with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is associated with age and smoking, but other determinants of the disease are incompletely understood. Clonal hematopoiesis of indeterminate potential (CHIP) is a common, age-related state in which somatic mutations in clonal blood populations induce aberrant inflammatory responses. Patients with CHIP have an elevated risk for cardiovascular disease, but the association of CHIP with COPD remains unclear. We analyzed whole-genome sequencing and whole-exome sequencing data to detect CHIP in 48 835 patients, of whom 8444 had moderate to very severe COPD, from four separate cohorts with COPD phenotyping and smoking history. We measured emphysema in murine models in which Tet2 was deleted in hematopoietic cells. In the COPDGene cohort, individuals with CHIP had risks of moderate-to-severe, severe, or very severe COPD that were 1.6 (adjusted 95% confidence interval [CI], 1.1-2.2) and 2.2 (adjusted 95% CI, 1.5-3.2) times greater than those for noncarriers. These findings were consistently observed in three additional cohorts and meta-analyses of all patients. CHIP was also associated with decreased FEV1% predicted in the COPDGene cohort (mean between-group differences, -5.7%; adjusted 95% CI, -8.8% to -2.6%), a finding replicated in additional cohorts. Smoke exposure was associated with a small but significant increased risk of having CHIP (odds ratio, 1.03 per 10 pack-years; 95% CI, 1.01-1.05 per 10 pack-years) in the meta-analysis of all patients. Inactivation of Tet2 in mouse hematopoietic cells exacerbated the development of emphysema and inflammation in models of cigarette smoke exposure. Somatic mutations in blood cells are associated with the development and severity of COPD, independent of age and cumulative smoke exposure.

SETD2 alterations impair DNA damage recognition and lead to resistance to chemotherapy in leukemia.

Mutations in SETD2, encoding the histone 3 lysine 36 trimethyltransferase, are enriched in relapsed acute lymphoblastic leukemia and MLL-rearranged acute leukemia. We investigated the impact of SETD2 mutations on chemotherapy sensitivity in isogenic leukemia cell lines and in murine leukemia generated from a conditional knockout of Setd2. SETD2 mutations led to resistance to DNA-damaging agents, cytarabine, 6-thioguanine, doxorubicin, and etoposide, but not to a non-DNA damaging agent, l-asparaginase. H3K36me3 localizes components of the DNA damage response (DDR) pathway and SETD2 mutation impaired DDR, blunting apoptosis induced by cytotoxic chemotherapy. Consistent with local recruitment of DDR, genomic regions with higher H3K36me3 had a lower mutation rate, which was increased with SETD2 mutation. Heterozygous conditional inactivation of Setd2 in a murine model decreased the latency of MLL-AF9-induced leukemia and caused resistance to cytarabine treatment in vivo, whereas homozygous loss delayed leukemia formation. Treatment with JIB-04, an inhibitor of the H3K9/36me3 demethylase KDM4A, restored H3K36me3 levels and sensitivity to cytarabine. These findings establish SETD2 alteration as a mechanism of resistance to DNA-damaging chemotherapy, consistent with a local loss of DDR, and identify a potential therapeutic strategy to target SETD2-mutant leukemias.

Physiologic Expression of Sf3b1(K700E) Causes Impaired Erythropoiesis, Aberrant Splicing, and Sensitivity to Therapeutic Spliceosome Modulation.

More than 80% of patients with the refractory anemia with ring sideroblasts subtype of myelodysplastic syndrome (MDS) have mutations in Splicing Factor 3B, Subunit 1 (SF3B1). We generated a conditional knockin mouse model of the most common SF3B1 mutation, Sf3b1(K700E). Sf3b1(K700E) mice develop macrocytic anemia due to a terminal erythroid maturation defect, erythroid dysplasia, and long-term hematopoietic stem cell (LT-HSC) expansion. Sf3b1(K700E) myeloid progenitors and SF3B1-mutant MDS patient samples demonstrate aberrant 3' splice-site selection associated with increased nonsense-mediated decay. Tet2 loss cooperates with Sf3b1(K700E) to cause a more severe erythroid and LT-HSC phenotype. Furthermore, the spliceosome modulator, E7017, selectively kills SF3B1(K700E)-expressing cells. Thus, SF3B1(K700E) expression reflects the phenotype of the mutation in MDS and may be a therapeutic target in MDS.

Role of casein kinase 1A1 in the biology and targeted therapy of del(5q) MDS.

The casein kinase 1A1 gene (CSNK1A1) is a putative tumor suppressor gene located in the common deleted region for del(5q) myelodysplastic syndrome (MDS). We generated a murine model with conditional inactivation of Csnk1a1 and found that Csnk1a1 haploinsufficiency induces hematopoietic stem cell expansion and a competitive repopulation advantage, whereas homozygous deletion induces hematopoietic stem cell failure. Based on this finding, we found that heterozygous inactivation of Csnk1a1 sensitizes cells to a CSNK1 inhibitor relative to cells with two intact alleles. In addition, we identified recurrent somatic mutations in CSNK1A1 on the nondeleted allele of patients with del(5q) MDS. These studies demonstrate that CSNK1A1 plays a central role in the biology of del(5q) MDS and is a promising therapeutic target.

Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing.

Genome sequencing studies have shown that human malignancies often bear mutations in four or more driver genes, but it is difficult to recapitulate this degree of genetic complexity in mouse models using conventional breeding. Here we use the CRISPR-Cas9 system of genome editing to overcome this limitation. By delivering combinations of small guide RNAs (sgRNAs) and Cas9 with a lentiviral vector, we modified up to five genes in a single mouse hematopoietic stem cell (HSC), leading to clonal outgrowth and myeloid malignancy. We thereby generated models of acute myeloid leukemia (AML) with cooperating mutations in genes encoding epigenetic modifiers, transcription factors and mediators of cytokine signaling, recapitulating the combinations of mutations observed in patients. Our results suggest that lentivirus-delivered sgRNA:Cas9 genome editing should be useful to engineer a broad array of in vivo cancer models that better reflect the complexity of human disease.

Csnk1a1 inhibition has p53-dependent therapeutic efficacy in acute myeloid leukemia.

Despite extensive insights into the underlying genetics and biology of acute myeloid leukemia (AML), overall survival remains poor and new therapies are needed. We found that casein kinase 1 α (Csnk1a1), a serine-threonine kinase, is essential for AML cell survival in vivo. Normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected by shRNA-mediated knockdown of Csnk1a1. To identify downstream mediators of Csnk1a1 critical for leukemia cells, we performed an in vivo pooled shRNA screen and gene expression profiling. We found that Csnk1a1 knockdown results in decreased Rps6 phosphorylation, increased p53 activity, and myeloid differentiation. Consistent with these observations, p53-null leukemias were insensitive to Csnk1a1 knockdown. We further evaluated whether D4476, a casein kinase 1 inhibitor, would exhibit selective antileukemic effects. Treatment of leukemia stem cells (LSCs) with D4476 showed highly selective killing of LSCs over normal HSPCs. In summary, these findings demonstrate that Csnk1a1 inhibition causes reduced Rps6 phosphorylation and activation of p53, resulting in selective elimination of leukemia cells, revealing Csnk1a1 as a potential therapeutic target for the treatment of AML.

In Vivo RNAi screening identifies a leukemia-specific dependence on integrin beta 3 signaling.

We used an in vivo small hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase Syk. In contrast, loss of Itgb3 in normal hematopoietic stem and progenitor cells did not affect engraftment, reconstitution, or differentiation. Finally, using an Itgb3 knockout mouse model, we confirmed that Itgb3 is dispensable for normal hematopoiesis but is required for leukemogenesis. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML.

Stress granules contribute to α-globin homeostasis in differentiating erythroid cells.

Hemoglobin is the major biosynthetic product of developing erythroid cells. Assembly of hemoglobin requires the balanced production of globin proteins and the oxygen-carrying heme moiety. The heme-regulated inhibitor kinase (HRI) participates in this process by phosphorylating eIF2α and inhibiting the translation of globin proteins when levels of free heme are limiting. HRI is also activated in erythroid cells subjected to oxidative stress. Phospho-eIF2α-mediated translational repression induces the assembly of stress granules (SG), cytoplasmic foci that harbor untranslated mRNAs and promote the survival of cells subjected to adverse environmental conditions. We have found that differentiating erythroid, but not myelomonocytic or megakaryocytic, murine and human progenitor cells assemble SGs, in vitro and in vivo. Targeted knockdown of HRI or G3BP, a protein required for SG assembly, inhibits spontaneous and arsenite-induced assembly of SGs in erythroid progenitor cells. This is accompanied by reduced α-globin production and increased apoptosis suggesting that G3BP+ SGs facilitate the survival of developing erythroid cells.

Densely interconnected transcriptional circuits control cell states in human hematopoiesis.

Though many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly coexpressed genes, some of which are restricted to a single lineage but most of which are expressed at variable levels across multiple lineages. We found densely interconnected cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed.

Monoterpene metabolism. Cloning, expression, and characterization of menthone reductases from peppermint.

(-)-Menthone is the predominant monoterpene produced in the essential oil of maturing peppermint (Mentha x piperita) leaves during the filling of epidermal oil glands. This early biosynthetic process is followed by a second, later oil maturation program (approximately coincident with flower initiation) in which the C3-carbonyl of menthone is reduced to yield (-)-(3R)-menthol and (+)-(3S)-neomenthol by two distinct NADPH-dependent ketoreductases. An activity-based in situ screen, by expression in Escherichia coli of 23 putative redox enzymes from an immature peppermint oil gland expressed sequence tag library, was used to isolate a cDNA encoding the latter menthone:(+)-(3S)-neomenthol reductase. Reverse transcription-PCR amplification and RACE were used to acquire the former menthone:(-)-(3R)-menthol reductase directly from mRNA isolated from the oil gland secretory cells of mature leaves. The deduced amino acid sequences of these two reductases share 73% identity, provide no apparent subcellular targeting information, and predict inclusion in the short-chain dehydrogenase/reductase family of enzymes. The menthone:(+)-(3S)-neomenthol reductase cDNA encodes a 35,722-D protein, and the recombinant enzyme yields 94% (+)-(3S)-neomenthol and 6% (-)-(3R)-menthol from (-)-menthone as substrate, and 86% (+)-(3S)-isomenthol and 14% (+)-(3R)-neoisomenthol from (+)-isomenthone as substrate, has a pH optimum of 9.3, and K(m) values of 674 mum, > 1 mm, and 10 mum for menthone, isomenthone, and NADPH, respectively, with a k(cat) of 0.06 s(-1). The recombinant menthone:(-)-(3R)-menthol reductase has a deduced size of 34,070 D and converts (-)-menthone to 95% (-)-(3R)-menthol and 5% (+)-(3S)-neomenthol, and (+)-isomenthone to 87% (+)-(3R)-neoisomenthol and 13% (+)-(3S)-isomenthol, displays optimum activity at neutral pH, and has K(m) values of 3.0 mum, 41 mum, and 0.12 mum for menthone, isomenthone, and NADPH, respectively, with a k(cat) of 0.6 s(-1). The respective activities of these menthone reductases account for all of the menthol isomers found in the essential oil of peppermint. Biotechnological exploitation of these genes could lead to improved production yields of (-)-menthol, the principal and characteristic flavor component of peppermint.

Monoterpene double-bond reductases of the (-)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (-)-isopiperitenone reductase and (+)-pulegone reductase of peppermint.

Random sequencing of a peppermint essential oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes. Full-length acquisitions of each type were screened by functional expression in Escherichia coli using a newly developed in situ assay. cDNA clones encoding the monoterpene double-bond reductases (-)-isopiperitenone reductase and (+)-pulegone reductase were isolated, representing two central steps in the biosynthesis of (-)-menthol, the principal component of peppermint essential oil, and the first reductase genes of terpenoid metabolism to be described. The (-)-isopiperitenone reductase cDNA has an open reading frame of 942 nucleotides that encodes a 314 residue protein with a calculated molecular weight of 34,409. The recombinant reductase has an optimum pH of 5.5, and K(m) values of 1.0 and 2.2 microM for (-)-isopiperitenone and NADPH, respectively, with k(cat) of 1.3s(-1) for the formation of the product (+)-cis-isopulegone. The (+)-pulegone reductase cDNA has an open reading frame of 1026 nucleotides and encodes a 342 residue protein with a calculated molecular weight of 37,914. This recombinant reductase catalyzes the reduction of the 4(8)-double bond of (+)-pulegone to produce both (-)-menthone and (+)-isomenthone in a 55:45 ratio, has an optimum pH of 5.0, and K(m) values of 2.3 and 6.9 microM for (+)-pulegone and NADPH, respectively, with k(cat) of 1.8s(-1). Deduced sequence comparison revealed that these two highly substrate specific double-bond reductases show less than 12% identity. (-)-Isopiperitenone reductase is a member of the short-chain dehydrogenase/reductase superfamily and (+)-pulegone reductase is a member of the medium-chain dehydrogenase/reductase superfamily, implying very different evolutionary origins in spite of the similarity in substrates utilized and reactions catalyzed.