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RXRA active mutation
with bexarotene, 525 overlapped with those of RXRA DT448/9PP-transduced cells. Likewise, of 74 downregulated genes in RXRA WT cells treated with bexarotene, 28 over- lapped with those in RXRA DT448/9PP-transduced cells.
Gene ontology pathway analysis showed enrichment of lipid metabolism and immune system signaling and down- regulation of RNA processing (Online Supplementary Tables S1 and S2). Gene set enrichment analysis of a curated list of genes regulated during myeloid development26 indicated that treatment of RXRA WT cells with bexarotene and expression of RXRA DT448/9PP both lead to a myeloid dif- ferentiation phenotype (upregulation of “Up” genes and
A
BCD
downregulation of “Down” genes) compared to untreated WT cells (Figure 5E, F).
Within this set of differentially expressed genes, we eval- uated transcripts associated with cell surface proteins. Sixty-seven cell surface transcripts were differentially expressed, according to RNA sequencing, between RXRA WT cells versus RXRA WT cells treated with bexarotene or RXRA DT448/9PP-transduced cells (Online Supplementary Figure S7A). As in the overall RNA sequencing analysis, these genes were associated with similar or greater upregu- lation in DT448/9PP-transduced cells than in the wild-type cells treated with bexarotene (Online Supplementary Figure
EFG
H
Figure 4. In vivo engraftment with RXRA WT cells versus RXRA DT448/9PP-transduced cells. (A) Experimental schema. KMT2A-MLLT3 WT leukemia cells were transduced with MSCV-RXRA WT-IRES-mCherry or MSCV-RXRA DT448/9PP- IRES-mCherry and transplanted immediately into sublethally irradiated mice. (B-D) Peripheral blood assessment at 4 weeks. WBC: white blood cells. (E) Assessment of proportion of mCherry+ cells before transplantation. (F) Assessment of mCherry in peripheral blood at 4 weeks. (G) Assessment of mCherry in bone marrow of moribund mice (of note, 2 RXRA DT448/9PP mice were found dead and were unable to be assessed and 1 was sacri- ficed but did not have splenomegaly). (H) Survival of mice after transplantation. *P<0.05, **P<0.01. The Mann-Whitney test was used in (B, F and G) with unequal variance. Otherwise, the t- test was used.
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