ALBERT

Description: The myelodysplastic syndromes (MDS) are common myeloid malignancies. Mutations in genes involved in pre-mRNA splicing (SF3B1, SRSF2, U2AF1 and ZRSR2) are the most common mutations found in MDS. There is evidence that some spliceosomal components play a role in the maintenance of genomic stability. Splicing is a transcription coupled process; splicing factor mutations affect transcription and may lead to the accumulation of R-loops (RNA-DNA hybrids with a displaced single stranded DNA). Mutations in the splicing factors SRSF2 and U2AF1 have been recently shown to increase R-loops formation in leukemia cell lines, resulting in increased DNA damage, replication stress and activation of the ATR-Chk1 pathway. SF3B1 is the most frequently mutated splicing factor gene in MDS, but a role for mutated SF3B1 in R-loop accumulation and DNA damage has not yet been reported in hematopoietic cells. We have investigated the effects of the common SF3B1 K700E mutation on R-loop formation and DNA damage response in MDS and leukemia cells. R-loop signals and the DNA damage response were measured by immunofluorescence staining using S9.6 and anti-γ-H2AX antibodies respectively. Firstly, we studied K562 (myeloid leukemia) cells with the SF3B1 K700E mutation and isogenic SF3B1 K700K wildtype (WT) K562 cells. K562 cells with SF3B1 mutation showed a significant increase in the number of S9.6 foci [Fold change (FC) 2.01, p

Description: A major recent discovery from large-scale sequencing studies was that over half of Myelodysplastic Syndromes (MDS) patients harbor mutations in splicing factor (SF) genes. SF mutations are the most common class of mutations in MDS and occur early in the course of the disease. These strongly suggest that SF mutations are key to the pathogenesis of MDS and can provide new therapeutic opportunities. However, identifying the downstream effects of SF mutations that are critical for the development of MDS presents a big challenge due to the cellular and genetic heterogeneity of primary patient samples, the unavailability of immortalized cell lines harboring SF mutations in the native genomic context and the limited conservation of alternative splicing isoforms between mice and humans. We previously showed that SF-mutant induced pluripotent stem cells (iPSCs) generated from MDS patients recapitulate key features of the disease upon differentiation into hematopoietic lineages, including cellular phenotypes (increased cell death, decreased clonogenicity and dysplastic morphology), sensitivity to splicing modulating drugs and the altered RNA binding specificity of mutant SFs (Chang et al. Stem Cell Reports, 2018). To further investigate the effects of SF mutations, we used CRISPR to introduce each of the 3 main canonical SF mutations (SRSF2 P95L, SF3B1 K700E, U2AF1 S34F) in the same normal iPSC line N-2.12 that we previously derived and extensively characterized in terms of pluripotency, genetic integrity and hematopoietic differentiation potential. The derivative iPSC lines contain the 3 SF mutations in isogenic conditions in the context of a diploid genome, in a heterozygous state, with both the normal and mutant alleles expressed at physiological and equal levels. To uncover potential new therapeutic targets and gain insights into the downstream effects of SF mutations, we set up CRISPR knockout (KO) lethality screens in hematopoietic progenitor cells (HPCs) derived from these SF-mutant iPSCs. We began with a gRNA library containing 224 gRNAs targeting 57 kinase genes (4 gRNAs per gene). The library was assembled and packaged in a lentiviral backbone also expressing GFP. Cas9 together with mCherry was expressed from a separate lentiviral vector. iPSCs were differentiated along the hematopoietic lineage, transduced on day 11, coinciding with the onset of the emergence of CD34+/CD45+ HPCs, and further cultured for up to day 27 to allow "dropout" of lethal genes, read out by next-generation sequencing (NGS). We titrated the lentiviral vectors to obtain transduction efficiency of nearly 100% for the Cas9 vector and up to 40-50% for the gRNA library (in order to obtain the highest percentage of cells harboring a single gRNA) in 500,000 HPCs, to ensure representation of the library of at least 500 cells per gRNA. To avoid population bottlenecks, we ensured that at least 500,000 cells were present in the culture at all times. All library gRNAs were present in the transduced cells and their distribution correlated tightly with that of the lentiviral supernatant. Technical repeats of independently prepared DNA samples, independent PCR reactions and independently generated NGS reads showed high reproducibility and absence of batch effects. The library was screened in 3 independent clones harboring each of the 3 canonical SF mutations, as well as in 3 clones of the parental normal line. This design allows the identification of potential convergent genes or pathways downstream of the 3 SF mutations and exclusion of non-synthetically lethal targets (which would also drop out in the isogenic normal cells). CRISPR scores were calculated as the average of the log of the final vs initial abundance of all gRNAs per gene, and showed a distribution consistent with the expectation that the majority of the gRNAs do not have a major impact on cell viability. Experiments with evidence of random genetic drifts from the CRISPR scores distribution were excluded from the analyses. Initial hits, defined as kinase genes with targeting gRNAs consistently depleted in SF-mutant lines of all 3 genotypes, but not in the normal isogenic cells are being validated with individual gene knockout and small molecule inhibition. In parallel, we are setting up CRISPR screens in expandable HPCs (eHPCs) derived from iPSCs. The latter can be expanded in culture for several weeks and could enable screening of larger or even genome-wide gRNA libraries. Disclosures No relevant conflicts of interest to declare.

Description: Myeloproliferative neoplasms (MPN) are characterized by the excessive production of one or more myeloid lineages and a propensity to progress to acute leukemia. In 2013, mutations in the CALR gene, encoding calreticulin, were identified in patients with MPN, mutually exclusive to the previously identified JAK2 and MPL (TPO-R) mutations. CALR mutations are frameshift mutations - typically a 52-bp deletion (type 1) or a 5-bp insertion (type 2) - that result in a novel C-terminus. The discovery of mutations in a ubiquitously expressed multifunctional protein like calreticulin was unanticipated. Subsequent studies found that CALR mutations lead to activation of JAK/STAT, mediated through aberrant interactions between mutant CALR and MPL, thus presenting an excellent opportunity for targeted therapy. However, the mechanism of MPL activation remains largely unexplained with prior studies using cell lines with exogenous expression of CALR and MPL following transfection. To create a more physiological cellular model to study the effects of CALR mutations, we established multiple iPSC lines from two patients with CALR-mutant MPN - one type 1-like (del34) and one type 2 (ins5) -, as well as from one patient with JAK2V617F MPN. All iPSC lines were confirmed to harbour the CALR or JAK2V617F mutation found in the corresponding patient, to express mutant calreticulin, as detected by flow cytometry using an antibody which specifically recognizes the novel calreticulin C-terminus, and to be karyotypically normal. Genetically matched iPSC lines with WT JAK2 could also be generated from the JAK2V617F (but not the CALR-mutant) patient cells in the same reprogramming round. CRISPR gene editing was used to generate isogenic CALR-corrected lines from both CALR-mutant patients. Furthermore, in order to facilitate biochemical studies, we used CRISPR to introduce a V5 epitope tag in one allele of the endogenous mutant or WT CALR gene, in mutant and isogenic corrected iPSC lines, respectively. We optimized an in vitro differentiation protocol for efficient derivation of megakaryocyte (MK) progenitors from iPSCs and found disease-relevant phenotypes, mainly TPO-independent MK colony formation in semi-solid media, which is the phenotypic hallmark of ex vivo primary MPN cells. In the absence of TPO, JAK2 V617F, CALR-mutant type 1-like and CALR-mutant type 2 iPSCs generated 52.1%, 58.7±22.2% and 59.8±3.6%, respectively, of the number of MK colonies generated in the presence of TPO, as opposed to 10%, 8.8±1.8% and 0.5±0.9%, respectively, for the matched WT JAK2, the corrected CALR-mutant type 1-like and the corrected CALR-mutant type 2 iPSCs. Isolated CALR mutant iPSC-derived CD41a+ MK progenitors had increased phosphorylation of STAT5 following cytokine starvation as compared to isogenic corrected and non-isogenic normal cells. CALR-mutant cells expressed equal transcript levels of the WT and mutant CALR alleles. However, mutant CALR protein levels were severely reduced, at levels 1~12% of those of the WT protein. This is consistent with previous studies documenting instability of mutant calreticulin. Transcriptomics (RNA-seq) and proteomics analyses of CD41a+-sorted MK progenitors derived from CALR mutant and isogenic corrected iPSCs are ongoing. These iPSC models offer the opportunity to study the effects of CALR mutations in a cellular context with both MPL and CALR (WT or mutant) expressed from their endogenous loci. They thus provide a powerful platform to investigate the disease mechanisms underlying CALR-mutant MPNs and to perform small molecule and genetic (CRISPR) screens to identify new therapeutic targets. Disclosures Iancu-Rubin: Merck: Research Funding; Incyte: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Merus: Research Funding; Formation Biologics: Research Funding; Janssen: Research Funding; Summer Road: Research Funding.

Description: Leukemia stem cells (LSCs) are believed to be a prominent source of relapse in acute myeloid leukemia (AML). Human AML LSCs are classically studied via xenotransplantation and defined as cells with (a) self-renewal ability, giving rise to leukemic engraftment that can be maintained over serial transplantation; and (b) ability to give rise to more differentiated progeny that are unable to engraft. LSCs are chemoresistant presumably because they have properties distinct from those of the bulk AML cells. A better understanding of the properties of LSCs can ultimately allow us to develop new therapies specifically targeting these cells to achieve lasting responses or even cures. We recently reported that induced pluripotent stem cells (iPSCs) generated from AML patients (AML-iPSCs) exhibit leukemic features upon hematopoietic differentiation, including extensive proliferation, maintenance of hematopoietic stem cell (HSC) markers, and serial engraftment of a lethal leukemia in immunodeficient mice (Kotini et al. Cell Stem Cell 2017). More recently, we found that the hematopoietic stem/progenitor cells (HSPCs) derived from AML-iPSCs exhibit phenotypic and functional heterogeneity. We first observed the presence of two cell populations with distinct growth characteristics and morphology: a population exhibiting adherent growth (adherent, A) and a population growing in suspension (S). The A cell population contained cells with immature morphology and HSC immunophenotype (CD34+/CD38-/CD90+/CD45RA-/ CD49f+), while the S fraction contained more differentiated cells (CD34-/low/CD38+/CD90-/CD45RA+). Experiments utilizing serial replating, single-cell plating of GFP-labeled cells and mathematical modeling revealed a hierarchical organization, whereby the A cells continuously give rise to the S cell fraction. Time-lapse imaging showed that A cells divide both symmetrically and asymmetrically with the majority (~60%) of cell divisions giving rise to two A cells and less frequently generating either one A and one S (~15%) or two S cells (~15%). Serial transplantation experiments in NSG mice revealed that the engraftment potential was largely contained within the A cell fraction. Cell cycle analysis showed that adherent cells contained a lower S and higher G0/G1 phase fraction than suspension cells. Thus, these AML-iPSC-derived hematopoietic cells exhibit hallmarks of an LSC model, namely phenotypic and functional heterogeneity and hierarchical organization, with the A fraction containing LSCs that serially transplant leukemia and give rise to more differentiated cells (S fraction) without engraftment potential. Transcriptome analyses showed that the A to S transition was predominantly mediated by gene upregulation with only 2 out of 418 differentially expressed genes being downregulated and revealed enrichment for HSC and LSC gene sets in the A fraction. Consistent with a general transcriptional upregulation characterizing the A to S transition, differentially accessible regions by ATAC-seq analysis were found predominantly in intragenic regions and enhancers in A cells, while they mostly localized in gene promoters in S cells. Time-course single cell-RNA-seq analyses and their integration with bulk RNA-seq and ATAC-seq data revealed cell clusters specific to the A cell fraction, which were enriched in HSC, LSC and hESC genes. These integrated analyses revealed candidate genes with a role in maintaining LSC properties. The transcription factor RUNX1 was particularly prominent, with RUNX1 motifs found highly accessible in A compared to S cells. Overexpression of RUNX1 in S cells conferred a more LSC-like immunophenotype. RUNX1 is a key transcriptional regulator of hematopoiesis. It has a well-characterized role during development of the hematopoietic system and is often mutated or translocated in AML. A role of RUNX1 in LSC maintenance was unanticipated in view of its known tumor-suppressor function. In further support for a role for RUNX1 in LSCs, we found RUNX1 expression to significantly correlate with survival in AML patient cohorts. In summary, we developed a new model that enables us to prospectively isolate large numbers of genetically clonal human AML LSCs and perform genome-wide integrative molecular studies, with which we obtained new insights into the biology of AML LSCs. Disclosures No relevant conflicts of interest to declare.

Description: Acute Myeloid Leukemia (AML) develops through the stepwise acquisition of mutations by hematopoietic stem or progenitor cells in a process of clonal evolution, which drives disease initiation, progression and relapse. In recent years, large scale sequencing of patients with AML yielded nearly complete catalogues of the driver mutations, uncovered their temporal occurrences and classified AML subgroups based on mutational profiles. The next daunting challenge is to understand how these mutations contribute to the development of Myelodysplastic Syndrome (MDS) and AML and how the order of their acquisition impacts disease progression. To study the individual and cooperative effects of driver mutations in MDS and AML and model clonal evolution, we developed iPSC models by introducing mutations in a stepwise manner through serial CRISPR/Cas9-mediated gene editing. We selected two mutational paths, representing de novo AML and secondary AML from preexisting MDS, respectively: (a) the combination DNMT3AR882H, NPM1c and FLT3ITD, the most common 3-gene co-mutation in AML, associated with poor prognosis; and (b) the combination SRSF2P95L, ASXL1 C-terminus truncation and NRASG12D, which represents the "chromatin-spliceosome" AML genomic classification group. The normal iPSC line N-2.12, previously extensively characterized in terms of pluripotency, genetic integrity and hematopoietic differentiation potential, was used as the parental line. Each gene editing step entailed screening of single cell clones by RFLP analysis, confirmation of correct targeting and presence of an intact untargeted allele by DNA sequencing, a second round of single-cell cloning to ensure clonality and karyotyping to exclude chromosomal abnormalities. At least two clones from each step were subjected to hematopoietic differentiation to test for potential phenotypic discrepancies and one representative clone was selected for the next gene editing step. For the DNMT3AR882H, FLT3ITD, SRSF2P95L and NRASG12D mutations, CRISPR-mediated homology directed repair (HDR) was used. ASXL1mutant lines were generated by CRISPR/Cas9 targeting of the beginning of exon 12 of ASXL1 and selection of clones with frame-shifting indels. NPM1c was introduced with a dox-inducible lentiviral vector. We thus derived a panel of single, double and triple mutant clones representing each step of the clonal evolution of AML.Single mutant clones (DNMT3AR882H and ASXL1-truncated) had completely normal hematopoietic differentiation potential (similar to that of the parental normal line and other non-isogenic normal lines), which is not surprising given that both these mutations are also found in individuals with clonal hematopoiesis without any hematopoietic defects. The double mutant SRSF2P95L-ASXL1 cells showed moderately impaired early hematopoietic differentiation potential with colony-forming ability reduced to approximately 50% of normal. In contrast, both triple mutant lines (DNMT3AR882H-FLT3ITD-NPM1c and SRSF2P95L-ASXL1-NRASG12D) showed a more severe differentiation block with markedly reduced colony-forming ability and prolonged growth in culture for over 11 weeks, in striking contrast to normal iPSC-derived hematopoietic progenitor cells (HPCs) which completely arrest their proliferation after 4 weeks. Transplantation of SRSF2P95L-ASXL1-NRASG12DHPCs into NSG mice resulted in detectable engraftment after 9 weeks in all transplanted animals. In conclusion, we successfully employed CRISPR/Cas to introduce driver mutations into iPSCs in a stepwise manner to "de novo" reconstruct the development of AML. These panels of iPSCs capture the distinct mutational steps along the evolution of AML in a clonal state and isogenic conditions. They should enable mechanistic studies into the processes of leukemogenesis and the effects of specific mutations acting at distinct stages of this progression, their cooperation and the order by which they are acquired. They should also prove a powerful tool to investigate the minimal genetic requirements for leukemia development, the potential need for cooperating epigenetic insults and the effects of the cellular context in myeloid transformation. Disclosures No relevant conflicts of interest to declare.