Hyperactivation of rat sarcoma (RAS) is common in human cancer. 1 As RAS proteins are difficult to target, identification of alternative means to block RAS oncogenic signaling is critical for developing cancer therapies. 2 We have previously shown that palmitoylation of neuroblastoma RAS (NRAS), which is predominantly mutated in human hematological malignances, 3 is essential for its leukemogenic activity, suggesting that targeting RAS palmitoylation may be an effective therapy at least for NRAS-related cancers. 4 S-palmitoylation is catalyzed by palmitoylacyltransferases (PATs). 5 To date, at least 23 mammalian PATs have been identified, a protein motif containing aspartate-histidinehistidine-cysteine residues (DHHC) conserved motif embedded in a cysteine-rich domain. 6 Zinc-finger DHHC-type containing 9 (ZDHHC9, a transmembrane protein) has been shown to function as a mammalian PAT with specificity for H-and NRAS in vitro. 7 In this study, we investigated the role of Zdhhc9 in modification of Nras in vivo, as well as its roles in normal hematopoiesis and in the development of leukemias induced by oncogenic Nras. A Zdhhc9-knockout (Zdhhc9KO) mouse strain was generated by using the strategy depicted in Supplementary Figure S1a. The Zdhhc9KO mice were born at the expected Mendelian ratio, and were viable, fertile and exhibited no noticeable abnormalities compared with their wild-type littermates during a follow-up period of 2 years (data not shown). Genotyping of mouse-tail genomic DNA shows that heterozygote mouse contains both wild type and mutant alleles of Zdhhc9, whereas mutant male mouse contains only the Zdhhc9 knockout allele (Supplementary Figure S1b). To study the effect of Zdhhc9 inactivation in hematopoietic system, we first confirmed that the expression of the Zdhhc9 transcripts was undetectable in hematopoietic tissues from Zdhhc9KO mice (Supplementary Figure S1c). Western blot analysis shows that the Zdhhc9 protein was not expressed in Zdhhc9KO mouse bone marrow (BM) and spleen (Figure 1a). To determine whether Zdhhc9 is a bona fide PAT for Ras palmitoylation in vivo, we performed the acyl-biotinyl exchange assay8 to assess the palmitoylation status of Ras in BM cells from Zdhhc9KO mice and the wild-type control mice. The results show that the palmitoylation level of Ras is significantly decreased, yet not abolished, in BM of Zdhhc9KO mice, comparing with their wildtype counterparts (Figure 1b). This result demonstrates that Zdhhc9 is a Ras PAT in vivo, but it is not the only one. As Zdhhc9 inactivation abrogates palmitoylation of the endogenous Ras proteins in Zdhhc9-deficient BM cells, we went on to evaluate the role of Zdhhc9 in normal hematopoiesis in vivo. We found that the absolute numbers of leukocytes, erythrocytes, platelets and hemoglobin level in peripheral blood of Zdhhc9KO mice are similar to that of wild-type control mice in the 6-months observation period (Figure 1c and Supplementary Figure S2a). The body weight and sizes of hematopoietic tissues—liver, spleen and thymus, are not affected by Zdhhc9 deficiency (Supplementary Figure S2b). The absolute numbers of cells in BM and spleen are also similar between Zdhhc9KO mice and the wild-type control mice (Supplementary Figure S2c). Interestingly, under stress conditions, the recovery of peripheral leukocytes was slightly quicker in single dose 5-fluorouracil treated Zdhhc9KO mice than that of Zdhhc9WT mice (Figure 1d). Moreover, the reduction of peripheral leukocyte counts and death of two doses 5-fluorouracil treated Zdhhc9KO mice were also moderately but significantly slower than that of wild-type mice …