Abstract:Despite the absence of mutations in the DNA repair machinery in myeloid malignancies, the advent of high-throughput sequencing and discovery of splicing and epigenetics defects in chronic myelomonocytic leukaemia (CMML) prompted us to revisit a pathogenic role for genes involved in DNA damage response. We screened for misregulated DNA repair genes by enhanced RNA-sequencing on bone marrow from a discovery cohort of 27 CMML patients and 9 controls. We validated 4 differentially expressed candidates in CMML CD34… Show more
“…We also found up-regulation of several genes unique to subsets of patients e.g. CDKN1A was up-regulated in a subset of patients’ stem cells, as observed previously by targeted q-RT-PCR analysis [11] . Fig.…”
Section: Resultssupporting
confidence: 88%
“…More broadly, we identify pathways aberrantly operational in stem cells from subsets of patients, including JAK-STAT signalling, inflammatory response, translation elongation and cell cycle progression, each of which are increasingly amenable to therapeutic modulation with novel agents. Aberrant expression of genes involved in inflammation and cell cycle was also observed in whole unsorted BM mononuclear cells and in monocytes from CMML patients, indicating that these altered regulatory networks are programmed within primitive stem cells and propagated throughout differentiation [ 11 , 13 ]. Activation of JAK-STAT pathway, without necessarily a demonstrable JAK2 mutation and particularly as a hypersensitive response to native GM-CSF, has been reported as a key feature of CMML, and drugs targeting this pathway have shown promise in both preclinical models and early phase clinical trials [44] , [45] , [46] , [47] , [48] .…”
Section: Discussionmentioning
confidence: 99%
“…While the transcriptome of CMML monocytes and bulk BM cells has been previously evaluated, the malignant stem cells in CMML have not been directly studied at the genome-wide level [11] , [12] , [13] , [14] , [15] . The Lin − CD34 + CD38 − BM compartment represents the apex of the haematopoietic hierarchy, typically representing <5% of total CD34 + cells and <0.25% of total BM MNCs [16] .…”
Background
Chronic myelomonocytic leukaemia (CMML) is a clinically heterogeneous stem cell malignancy with overlapping features of myelodysplasia and myeloproliferation. Over 90% of patients carry mutations in epigenetic and/or splicing genes, typically detectable in the Lin
−
CD34
+
CD38
−
immunophenotypic stem cell compartment in which the leukaemia-initiating cells reside. Transcriptional dysregulation at the stem cell level is likely fundamental to disease onset and progression.
Methods
We performed single-cell RNA sequencing on 6826 Lin
−
CD34
+
CD38
−
stem cells from CMML patients and healthy controls using the droplet-based, ultra-high-throughput 10x platform.
Findings
We found substantial inter- and intra-patient heterogeneity, with CMML stem cells displaying distinctive transcriptional programs. Compared with normal controls, CMML stem cells exhibited transcriptomes characterized by increased expression of myeloid-lineage and cell cycle genes, and lower expression of genes selectively expressed by normal haematopoietic stem cells. Neutrophil-primed progenitor genes and a MYC transcription factor regulome were prominent in stem cells from CMML-1 patients, whereas CMML-2 stem cells exhibited strong expression of interferon-regulatory factor regulomes, including those associated with IRF1, IRF7 and IRF8. CMML-1 and CMML-2 stem cells (stages distinguished by proportion of downstream blasts and promonocytes) differed substantially in both transcriptome and pseudotime, indicating fundamentally different biology underpinning these disease states. Gene expression and pathway analyses highlighted potentially tractable therapeutic vulnerabilities for downstream investigation. Importantly, CMML patients harboured variably-sized subpopulations of transcriptionally normal stem cells, indicating a potential reservoir to restore functional haematopoiesis.
Interpretation
Our findings provide novel insights into the CMML stem cell compartment, revealing an unexpected degree of heterogeneity and demonstrating that CMML stem cell transcriptomes anticipate disease morphology, and therefore outcome.
Funding
Project funding was supported by Oglesby Charitable Trust, Cancer Research UK, Blood Cancer UK, and UK Medical Research Council.
“…We also found up-regulation of several genes unique to subsets of patients e.g. CDKN1A was up-regulated in a subset of patients’ stem cells, as observed previously by targeted q-RT-PCR analysis [11] . Fig.…”
Section: Resultssupporting
confidence: 88%
“…More broadly, we identify pathways aberrantly operational in stem cells from subsets of patients, including JAK-STAT signalling, inflammatory response, translation elongation and cell cycle progression, each of which are increasingly amenable to therapeutic modulation with novel agents. Aberrant expression of genes involved in inflammation and cell cycle was also observed in whole unsorted BM mononuclear cells and in monocytes from CMML patients, indicating that these altered regulatory networks are programmed within primitive stem cells and propagated throughout differentiation [ 11 , 13 ]. Activation of JAK-STAT pathway, without necessarily a demonstrable JAK2 mutation and particularly as a hypersensitive response to native GM-CSF, has been reported as a key feature of CMML, and drugs targeting this pathway have shown promise in both preclinical models and early phase clinical trials [44] , [45] , [46] , [47] , [48] .…”
Section: Discussionmentioning
confidence: 99%
“…While the transcriptome of CMML monocytes and bulk BM cells has been previously evaluated, the malignant stem cells in CMML have not been directly studied at the genome-wide level [11] , [12] , [13] , [14] , [15] . The Lin − CD34 + CD38 − BM compartment represents the apex of the haematopoietic hierarchy, typically representing <5% of total CD34 + cells and <0.25% of total BM MNCs [16] .…”
Background
Chronic myelomonocytic leukaemia (CMML) is a clinically heterogeneous stem cell malignancy with overlapping features of myelodysplasia and myeloproliferation. Over 90% of patients carry mutations in epigenetic and/or splicing genes, typically detectable in the Lin
−
CD34
+
CD38
−
immunophenotypic stem cell compartment in which the leukaemia-initiating cells reside. Transcriptional dysregulation at the stem cell level is likely fundamental to disease onset and progression.
Methods
We performed single-cell RNA sequencing on 6826 Lin
−
CD34
+
CD38
−
stem cells from CMML patients and healthy controls using the droplet-based, ultra-high-throughput 10x platform.
Findings
We found substantial inter- and intra-patient heterogeneity, with CMML stem cells displaying distinctive transcriptional programs. Compared with normal controls, CMML stem cells exhibited transcriptomes characterized by increased expression of myeloid-lineage and cell cycle genes, and lower expression of genes selectively expressed by normal haematopoietic stem cells. Neutrophil-primed progenitor genes and a MYC transcription factor regulome were prominent in stem cells from CMML-1 patients, whereas CMML-2 stem cells exhibited strong expression of interferon-regulatory factor regulomes, including those associated with IRF1, IRF7 and IRF8. CMML-1 and CMML-2 stem cells (stages distinguished by proportion of downstream blasts and promonocytes) differed substantially in both transcriptome and pseudotime, indicating fundamentally different biology underpinning these disease states. Gene expression and pathway analyses highlighted potentially tractable therapeutic vulnerabilities for downstream investigation. Importantly, CMML patients harboured variably-sized subpopulations of transcriptionally normal stem cells, indicating a potential reservoir to restore functional haematopoiesis.
Interpretation
Our findings provide novel insights into the CMML stem cell compartment, revealing an unexpected degree of heterogeneity and demonstrating that CMML stem cell transcriptomes anticipate disease morphology, and therefore outcome.
Funding
Project funding was supported by Oglesby Charitable Trust, Cancer Research UK, Blood Cancer UK, and UK Medical Research Council.
“…7,8 Targeted mutation (TruSight Myeloid Sequencing Panel, Illumina) and transcriptome libraries for RNA sequencing 8 were generated and sequenced on an Illumina HiSeq sequencer (Data S1). Four independent validation cohorts (VCs) with CMML patients treated at Murcia (VC1, n = 25), 9 NTUH (VC2, n = 24), the Christie Hospital (VC3, n = 14) and the Mayo Clinic (VC4, n = 20) 10 were included. The NTUH Research Ethics Committee approved the study (#201709072RINC).…”
Summary
Leukaemic stem cell (LSC) gene expression has recently been linked to prognosis in patients with acute myeloid leukaemia (17‐gene LSC score, LSC‐17) and myelodysplastic syndromes. Although chronic myelomonocytic leukaemia (CMML) is regarded as a stem cell disorder, the clinical and biological impact of LSCs on CMML patients remains elusive. Making use of multiple independent validation cohorts, we here describe a concise three‐gene expression signature (LSC‐3, derived from the LSC‐17 score) as an independent and robust prognostic factor for leukaemia‐free and overall survival in CMML. We propose that LSC‐3 could be used to supplement existing risk stratification systems, to improve prognostic performance and guide management decisions.
“…To understand the landscape of mis-splicing in SRSF2 mutated cells, we literature curated mis-spliced genes from 16 different human and murine datasets of SRSF2 mutation, including the Cas9 Srsf2 cells described above (Supplemental Table 1) [3][4][5]18,[32][33][34][35][36] . Metascape pathway enrichment analysis was performed on genes that were identified in more than 4 of the 16 datasets, regardless of the type of mis-spliced event 25 .…”
Section: Cell Cycle Regulation and Dna Repair Pathways Are Over-represented Within The Mis-spliced Transcript Set Of Srsf2 Mutant Cellsmentioning
Current strategies to target RNA splicing mutant myeloid cancers proposes targeting the remaining splicing apparatus. This approach has only been modestly sensitizing and is also toxic to non-mutant bearing wild-type cells. To explore potentially exploitable genetic interactions with spliceosome mutations, we combined data mining and functional screening for synthetic lethal interactions with an Srsf2P95H/+ mutation. Analysis of mis-splicing events in a series of both human and murine SRSF2P95H mutant samples across multiple myeloid diseases (AML, MDS, CMML) was performed to identify conserved mis-splicing events. From this analysis, we identified that the cell cycle and DNA repair pathways were overrepresented within the conserved mis-spliced transcript sets. In parallel, to functionally define pathways essential for survival and proliferation of Srsf2P95H/+ cells, we performed a genome-wide CRISPR loss of function screen using Hoxb8 immortalized R26-CreERki/+ Srsf2P95H/+ and R26-CreERki/+ Srsf2+/+ cell lines. We assessed loss of sgRNA representation at three timepoints: immediately after Srsf2P95H/+ activation, and at one week and two weeks post Srsf2P95H/+ mutation. Pathway analysis demonstrated that the cell cycle and DNA damage response pathways were amongst the top synthetic lethal pathways with Srsf2P95H/+ mutation. Based on the loss of guide RNAs targeting Cdk6, we identified that Palbociclib, a CDK6 inhibitor, showed preferential sensitivity in Srsf2P95H/+ cell lines and in primary non-immortalized lin-cKIT+Sca-1+ cells compared to wild type controls. Our data strongly suggest that the cell cycle and DNA damage response pathways are required for Srsf2P95H/+ cell survival, and that Palbociclib could be an alternative therapeutic option for targeting SRSF2 mutant cancers.
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