Page 119 - Haematologica Vol. 110 - January 2025

ARTICLE - LIPA modulates venetoclax/TKI response in bpCML M. Minhajuddin et al.
venetoclax/dasatinib combination. (C, D) Gene set enrichment analysis showing enrichment of genes related to lysosome biolo- gy in venetoclax/dasatinib-treated LSC. (E) Leukemia cells (GY+; lin–) were pretreated with bafilomycin (10 nM) for 1 h and then treated in vitro for 24 h with venetoclax alone (100 nM), dasatinib alone (100 nM), or their combination. Cell viability was measured compared to that of cells treated with the vehicle control. (F) Heatmap showing expression of representative lysosome-related genes in LSC untreated versus treated with the venetoclax/dasatinib combination. (G) LIPA expression in response to dasatinib alone and the venetoclax/dasatinib combination. Error bars denote mean ± standard deviation from triplicate experiments. Sta- tistical analyses were performed using a Student t test. NS: not statistically significant, ***P≤0.001, ****P≤0.0001. d: day; Com- bo: venetoclax/dasatinib combination; BM: bone marrow; RNA-seq: RNA sequencing; PCA: principal component analysis; Ven: venetoclax; Dasa: dasatinib; KEGG: Kyoto Encyclopedia of Genes and Genomes; NES: normalized enrichment score; GSEA: gene set enrichment analysis; FDR: false discovery rate; Baf: bafilomycin.
venetoclax and/or dasatinib strongly increased sensitivity to the ven/dasa treatment, implying a protective role for lysosome activity. Of the genes that were upregulated in the lysosome pathway specifically due to treatment with both venetoclax and dasatinib, we found that lysosomal acid lipase (LIPA or LAL) was consistently elevated (Figure 4F, G). This enzyme is also referred to as cholesterol ester hydrolase and is involved in hydrolysis and recycling of cholesterol and free fatty acids for cellular energy.23 The upregulation of LIPA in the ven/dasa-treated LSC population was confirmed by quantitative polymerase chain reaction analysis (Online Supplementary Figure S4I).
Metabolomic analysis of GY cells treated with venetoclax plus dasatinib reveals upregulation of free fatty acids
Since LIPA is known to positively regulate free fatty acid levels, we performed metabolomic analyses of LSC iso- lated after 4 h of treatment with ven/dasa (Figure 6A). We found that several fatty acids were upregulated in the ven/ dasa-treated group compared to control (Figure 6B). More importantly, supplementing the medium with these same fatty acids (e.g., a-linolenic acid and dihomo-g-linolenic acid) resulted in partial rescue from ven/dasa-mediated cell death, implicating upregulation of fatty acid levels as a protective response to ven/dasa challenge (Figure 6C, D).
Modulation of LIPA expression results in altered response to venetoclax and dasatinib treatment in a mouse model of blast phase chronic myeloid leukemia To examine the role of endogenous LIPA in leukemia cells, we utilized CRISPR technology and electroporated Cas9-sgRNA complexes into murine GY leukemia cells to knockout LIPA.36 As shown in Figure 7A, B, this approach achieved strong knockdown of LIPA at both the mRNA and protein levels. Treatment of LIPA knockout cells with ven/dasa resulted in increased sensitivity (Figure 7C), further corroborating the concept that upregulation of lysosomal activity serves as a protective response to ven/dasa challenge. We also overexpressed the gene in murine GY leukemia cells (Figure 7D, E) and found that treatment of LIPA-overexpressing leukemia cells with ven/dasa resulted in increased re- sistance compared to cells transduced with vector alone (Figure 7F). These data suggest that increased free fatty acids due to higher expression of LIPA results in diminished
cytotoxicity of ven/dasa. This implies that utilization of fatty acids by leukemic cells may be important for diminished drug response. Thus, we hypothesized that inhibiting the expression of carnitine palmitoyltransferase 1A (CPT1A), an important free fatty acid mitochondrial membrane trans- porter, would be able to modulate the response to ven/ dasa. Indeed, knockdown of CPT1A using siRNA (Figure 7G) resulted in increased sensitivity of the GY cells to ven/da- sa treatment compared to that of cells transfected with scrambled siRNA (Figure 7H).
Modulation of LIPA expression results in altered response to venetoclax and dasatinib treatment in human blast phase chronic myeloid leukemia samples To determine whether the findings from GY mouse cells were also evident in human bpCML cells, we examined the effect of both LIPA knockdown and CPT1A knockdown in primary human bpCML using an siRNA approach. As shown in Figure 8A, B and Online Supplementary Figure S4C, D, we confirmed significant knockdown of LIPA expression at mRNA and protein levels and found that treatment of LIPA knockdown cells with ven/dasa resulted in increased drug sensitivity compared to that of scrambled control cells (Figure 8C, Online Supplementary Figure S4E). Furthermore, after confirming knockdown of CPT1A expression in primary human samples (Figure 8D, Online Supplementary Figure S4F, G) we similarly demonstrated increased sensitivity to ven/dasa compared to that of scrambled control cells (Figure 8E, Online Supplementary Figure S4H). Together these results suggest that treatment with ven/dasa results in upregulation of genes associated with lysosome biology, in particular LIPA, as a protective response. LIPA is known to increase production of free fatty acids that can be used as a fuel source, which in part may be responsible for the diminished response to ven/dasa.
Discussion
The primary goal of this study was to investigate the mo- lecular mechanisms that mediate the LSC-targeting ac- tivity of venetoclax in combination with a TKI in bpCML. In agreement with the studies of Carter et al.,21 we observed that venetoclax in combination with a TKI is a highly effec- tive LSC-targeting regimen in both xenograft studies using
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