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. 2022 May 28;14(11):2681.
doi: 10.3390/cancers14112681.

Pitavastatin Is Anti-Leukemic in a Bone Marrow Microenvironment Model of B-Lineage Acute Lymphoblastic Leukemia

Debbie Piktel  1 , Rajesh R Nair  1 , Stephanie L Rellick  1 , Werner J Geldenhuys  2 , Karen H Martin  1   3 , Michael D Craig  4 , Laura F Gibson  1   3
Affiliations

Pitavastatin Is Anti-Leukemic in a Bone Marrow Microenvironment Model of B-Lineage Acute Lymphoblastic Leukemia

Debbie Piktel et al. Cancers (Basel). .

Abstract

The lack of complete therapeutic success in the treatment of B-cell acute lymphoblastic leukemia (ALL) has been attributed, in part, to a subset of cells within the bone marrow microenvironment that are drug resistant. Recently, the cholesterol synthesis inhibitor, pitavastatin (PIT), was shown to be active in acute myeloid leukemia, prompting us to evaluate it in our in vitro co-culture model, which supports a chemo-resistant ALL population. We used phospho-protein profiling to evaluate the use of lipid metabolic active compounds in these chemo-resistant cells, due to the up-regulation of multiple active survival signals. In a co-culture with stromal cells, a shift towards anabolic processes occurred, which was further confirmed by assays showing increased lipid content. The treatment of REH leukemia cells with pitavastatin in the co-culture model resulted in significantly higher leukemic cell death than exposure to the standard-of-care chemotherapeutic agent, cytarabine (Ara-C). Our data demonstrates the use of pitavastatin as a possible alternative treatment strategy to improve patient outcomes in chemo-resistant, relapsed ALL.

Keywords: ALL; drug resistance; lipid metabolism; metabolism.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Activation of survival signaling pathways in drug-resistant phase dim (PD) cells. (A) REH cells were co-cultured with BMSC, the S and PD ALL cells were isolated, and the phosphorylation of protein kinases was compared with the PD expression level compared to the S sub-population. (B) REH cells were co-cultured with BMSC and left untreated (control) or treated with MK-2206. The PD REH tumor cells cultures were isolated from the control and MK-2206 cultures, and the relative level of protein kinase phosphorylation was determined.
Figure 2
Figure 2
Drug-resistant phase dim (PD) cells are characterized by anabolic metabolism. REH cells were co-cultured with HOB, and the tumor cells in suspension (S) and the phase dim (PD) cells were isolated and compared to REH cells grown in media alone (M). (A) RT-PCR was used to determine the expression levels of GLUT-1, 3, and 4. Data is represented as mean ± SEM and is a representative of an experiment performed in triplicate at least two independent times. (B) The different cell populations were treated with a pan GLUT inhibitor (GLUTi II) or vehicle control followed by incubation with 2-NBDG. Flow cytometry was used to measure glucose content in cells shown as mean fluorescent intensity (MFI). (C) A glycogen colorimetric assay was used to measure the glycogen content in the isolated tumor cell populations. (D) Western blot analysis was used to measure the amount of AMPK and phospho-AMPK. The blots are representative of three independent experiments. * p < 0.05 when compared to cells grown in media alone. ** p < 0.05 when compared to suspension cells isolated from co-culture or tumor cells grown in media alone. The uncropped blots are shown in Supplementary Materials.
Figure 3
Figure 3
Drug-resistant phase dim (PD) cells have increased lipid content. REH cells were co-cultured with (HOB), and the cells in suspension (S) and the phase dim (PD) cells were isolated and compared to REH cells grown in media alone (M). Nile Red staining was completed to visualize lipid content. Scale bar = 20 μm. Red (Nile Red); Blue (DAPI nuclear stain).
Figure 4
Figure 4
Inhibition of lipid synthesis decreases ALL cell proliferation. REH cell lines were treated with the indicated concentrations of pitavastatin, and the cell proliferation was measured as described in the methods. The data are represented as mean ± SD of a study performed in triplicate at least three independent times.
Figure 5
Figure 5
Pitavastatin decreases ALL cell viability in co-culture. REH cells were grown in a co-culture with HOB and were treated with Ara-C (0.1 μM), BMS-303141 (BMS; 10 μM), pitavastatin (PIT; 1 μM) or a combination of BMS-141303 and pitavastatin (BMS + PIT). The cells in suspension (S) and the phase dim (PD) cells were isolated and compared to REH cells grown in media alone (M). The total cells and the live cell population (A) were used to calculate the % viability (B). The data is presented as mean ± SEM and is representative of a study performed in triplicate and conducted three independent times. * p < 0.05 when compared to vehicle/media treated.
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