In Vivo barcoded CRISPR-Cas9 Screen Identifies Ncoa4-mediated Ferritinophagy As a Dependence in Tet2-Deficient Haemopoiesis

Loss of function mutations in TET2 resulting in age-related expansion of mutant clones are commonly seen in individuals with clonal haemopoiesis (CH). Murine models of Tet2 knockout (KO) CH replicate this observation of increased haematopoietic stem progenitor cell (HSPC) fitness. We sought to identify regulators of Tet2 KO HSPC using an invivo CRISPR-Cas9 screen. Results of conventional in vivo CRISPR-Cas9 screens can be confounded by heterogeneity in physiological expansion of engrafted HSPCs post-transplant. To account for this, we encoded a DNA sequence as an unique molecular identifier (UMI) within the single guide RNA (sgRNA) expressing vector, enabling clonal tracing of each HSPC transduction event alongside identification of the corresponding sgRNA-mediated genetic perturbation.

Methods: Lineage-Sca1+cKit+ (LSK) HSPCs from Cas9-expressing Tet2 KO or Tet2 WT mice were transduced with a library of 12727 sgRNA targeting 2,055 genes (6 sgRNA/gene) and transplanted into lethally irradiated recipients. Recipient bone marrow was harvested after 14 weeks and sgRNA frequency (clone size) and number of UMI (clone number), was assessed. We subsequently validated candidate genes by CRISPR-Cas9 and transgenic mouse models. Mechanistic studies were undertaken using murine primary and Hoxb8-ER immortalised HSPC and primary human CD34+ cells.

Results: Our in vivo barcoded CRISPR-Cas9 screen identified candidate genes preferentially required for Tet2 KO clonal outgrowth as compared to WT. These included known oncogenic regulators of myeloid malignancies (e.g. Prmt5, Six1, Cdx2). Ncoa4 was also amongst the top candidates. We performed a secondary validation using a library of sgRNA in Cas9+CD150+CD48-haematopoietic stem cells (HSCs). This confirmed Ncoa4 KO reduced the number and size of Tet2 KO clones as compared to WT. Tet2 KO HSCs were reduced in HSPC transduced with Ncoa4 sgRNA as compared to non-targeting guide. Consistent with this, single cell RNA-seq demonstrated a decreased stem cell signature in Tet2 KO Cas9 LSK HSPCs transduced with a Ncoa4, as compared to control sgRNA. Using a Cre-conditional Tet2 KO with and without Ncoa4 KO, we demonstrated that expansion of Tet2 KO cells is reduced by concomitant Ncoa4 KO at 12 weeks in competitive transplantation (BMT). Ncoa4 targets ferritin (Fth1) for autophagic degradation, releasing iron from the lysosome intracellularly (Mancias et al, 2014). Following a Tet2 KO vs WT competitive BMT, mice treated with ironomycin, an inhibitor of lysosomal iron release, reduced Tet2 KO mediated growth advantage in myeloid and LSK compartments. RNA-seq demonstrated significant overlap in both up and downregulated genes in Tet2 KO Hoxb8 HSPCs treated either with Ncoa4 shRNA or ironomycin.

We hypothesised that an increased iron requirement in Tet2 KO HSPCs results in dependence on Ncoa4-mediated ferritinophagy. Consistent with this was increased intracellular free iron in Tet2 KO as compared to WT HSPCs. We confirmed that Fth1 flux in both WT and Tet2 KO cells is dependent on lysosomal degradation which is higher in Tet2 KO cells as compared to WT. In vitroTet2 KO cell growth advantage over WT is dependent on iron availability, which was rescued by ferric ammonium citrate. A major demand for intracellular iron is the mitochondria. Proteomic analysis demonstrated metal cluster binding enzymes were amongst the top gene set enriched, including components of the electron transport chain (ETC), in Tet2 KO vs WT Hoxb8 HSPCs. Consistent with this, transmission electron microscopy demonstrated increased mitochondrial cristae, where the ETC resides, in Tet2 KO as compared to WT LSK HSPCs. Tet2 KO Hoxb8 HSPC, as compared to WT, had increased copies of mitochondrial DNA, correlating with increased mitochondrial mass. Functional mitostress Seahorse assay demonstrated increased oxidative phosphorylation (OxPhos), but not glycolytic ATP production in Tet2 KO Hoxb8 HSPC as compared to WT, and in CRISPR-Cas9 TET2 KO as compared to safe-harbour edited (AAVS1) human CD34+ HSPCs. The increased OxPhos-ATP production in Tet2 KO cells was reduced either by Ncoa4 knockdown or ironomycin.

Conclusions: We developed and validated an in vivo CRISPR-Cas9 screening platform to identify new regulators of haemopoiesis. This system identified labile iron flux as an important regulator of Tet2 KO clonal expansion, in part due to the increased mitochondrial OxPhos demand in Tet2 KO HSPCs.

Armstrong:C4 Therapeutics: Other: Scientific Advisory Board; Neomorph Inc.: Other: Scientific Advisory Board; Syndax: Research Funding; Hyku Therapeutics: Consultancy; Accent Therapeutics: Other: Scientific Advisory Board; Nimbus Therapeutics: Consultancy; Janssen: Research Funding.