CD45 inhibition in myeloid leukaemia cells sensitizes cellular responsiveness to chemotherapy

Abstract

Myeloid malignancies are a group of blood disorders characterized by the proliferation of one or more haematopoietic myeloid cell lineages, predominantly in the bone marrow, and are often caused by aberrant protein tyrosine kinase activity. The protein tyrosine phosphatase CD45 is a trans-membrane molecule expressed on all haemopoietic blood cells except that of platelets and red cells. CD45 regulates various cellular physiological processes including proliferation, apoptosis, and lymphocyte activation. However, its role in chemotherapy response is still unknown; therefore, the aim of this study was to investigate the role of CD45 in myeloid malignancies in terms of cellular growth, apoptosis, and response to chemotherapy. The expression of CD45 on myeloid leukaemia primary cells and cell lines was heterogeneous with HEL and OCI-AML3 cells showing the highest level. Inhibition of CD45 resulted in increased cellular sensitivity to cytarabine and ruxolitinib, the two main therapies for AML and MPN. Bioinformatics analysis identified genes whose expression was correlated with CD45 expression such as JAK2, ACTR2, THAP3 Serglycin, and PBX-1 genes, as well as licensed drugs (alendronate, allopurinol, and balsalazide), which could be repurposed as CD45 inhibitors which effectively increases sensitivity to cytarabine and ruxolitinib at low doses. Therefore, CD45 inhibition could be explored as a potential therapeutic partner for treatment of myeloid malignancies in combination with chemotherapy such as cytarabine especially for elderly patients and those showing chemotherapy resistance.

Keywords: AML; CD45; Common leukocyte antigen (CLA); Myeloid leukaemia; PTPRC.

Fig. 1

Assessment of CD45 expression on myeloid leukaemia cells and their response to cytarabine. Western blot analysis of CD45 on cell lines (A). A scatter blot was performed to facilitate a clear comparison of results obtained by PCR, flow cytometry, and western blotting (B) showed OCI-AML3, and HEL cells have a high CD45 expression, while NALM-6 and UT-7 cells characterise low CD45 expression. Western blots of CD45 expression on cells from normal individuals, AML, and MPN patients (C), and further validation of ex vivo CD45 expression by RT-PCR (D). Cytarabine dose response curves on myeloid leukaemia cells (E). The error bar represents standard deviation of the mean from three repeats (n = 3, SEM); ex vivo experiments represent peripheral blood cells of normal individuals (14 samples), AML (18 samples), and MPN (8 samples) patients

Fig. 2

A Identification of genes whose expression is correlated with CD45 expression was performed by Partek Genomic Suite software. The genes then further analyzed by Quadratic software which results in visualization of a heatmap showed negative CD45 correlated genes, and another showed positive CD45 correlated genes. This ends with selection of three negative CD45 correlated genes, JAK2, THAP3, and ACTR2, and two positive CD45 correlated genes: PBX-1 and Serglycin. Heatmaps showed identification of a list of genes negatively correlated with CD45 expression, and another of positively correlated genes. B Assessment of optimization for non-toxic drug concentrations on normal, AML, and MPN myeloid primary cells and HEL, SKM, and OCI-AML3 cell lines. The drug concentrations used were validated to be non-toxic to the cells as alendronate (D), allopurinol (A), and balsalazide (B) were at 0.01 mM, cytarabine (C) at 0.01 µM, JAK inhibitor (JI) at 1 µM, interleukin 6 (IL-6) at 100 nM, and vanadate (V) at 50 µM. Data is based on three biological replicates and three technical replicates

Fig. 3

Validation of CD45 inhibitory effect of the selected drugs. CD45 inhibitor drugs at 0.01 mM showed efficacy to inhibit CD45 expression on HEL, SKM, and OCI-AML3 cell lines (A), and on primary myeloid leukaemia and normal cells (B). Western blot showed significant inhibition of CD45 (p-value < 0.0001) on MPN cells and AML cells (p-value < 0.004 but non-significant on normal cells confirmed by densitometry of the bands (C). Effect of CD45 inhibitor drugs on cellular response to cytarabine on cells treated with CD45 inhibitors (D). Abbreviations; D, alendronate; A, allopurinol; B, balsalazide. All experiments represent three independent biological replicates (n = 3). The error bar represents standard deviation of the mean from three repeats. Ex vivo experiments represent of peripheral blood cells of normal individuals (14 samples), AML (18 samples), and MPN (8 samples) patients

Fig. 4

A CD45 over-expression has no significant effect on cellular response to cytarabine. CAS9 + UT-7 and CAS9 + NALM-6 cell lines were treated with pre-designed CD45 DNA plasmid for 48 h followed by assessment of CD45 over-expression by PCR. CD45 over-expressed cells were then treated with a dose range of cytarabine. Another set of UT-7 and NALM-6 cells with untreated CD45 expression were introduced with the same range of cytarabine dose for comparison with cell viability being assessed using the Cell Titer-Glo® Assay at 48 and 72 h. B Network analysis with STRING software to identify any potential protein interactions. An edge was drawn with up to seven differently coloured lines, representing the existence of the seven types of evidence used for predicting the correlations: a red line marks the presence of fusion evidence, a yellow line represents text mining evidence, a purple line shows experimental evidence, a blue line indicates cooccurrence evidence, a light blue line indicates database evidence, a green line indicates neighbourhood evidence, and a black line represents co-expression evidence. C Influence of JAK2 inhibitor on CD45 expression was assessed by western blot on myeloid cells treated with 1.0 µM ruxolitinib (JAK inhibitor) and 0.01 mM of alendronate (D), allopurinol (A), and balsalazide (B) showed that JAK inhibitor alone did not affect CD45 expression, but when combined with any of CD45 inhibitors. D Validation of correlation between CD45 expression and the expression of the investigated genes. Western blot showed that expression of JAK2, ACTR2, and THAP3 genes was negative to CD45 expression, while PBX-1 and SEG genes were positive to CD45 expression All experiments represent three independent biological replicates (n = 3). The error bar represents standard deviation of the mean from three repeats. Ex vivo experiments represent of peripheral blood cells of normal individuals (14 samples), AML (18 samples), and MPN (8 samples) patients

Fig. 5

Drug combination experiments on myeloid leukaemia cells. Pair combinations of one CD45 inhibitor drug with IC₂₀ cytarabine resulted in a great synergism in HEL, SKM, and OCI-AML3 cells as well as primary cells from MPN and AML patients but were non-toxic to normal cells. Likewise, double drug combination of JAK inhibitor with CD45 inhibitor drugs was non-toxic to normal cells, but can kill HEL cells, MPN, and AML primary cells (A). Triple drug combination of two CD45 inhibitors with cytarabine worked synergistically on HEL, SKM, and OCI-AML3 cells as well as primary cells, but non-toxic to normal cells. Additionally, JAK inhibitor was strongly synergized with allopurinol and cytarabine (A + JI + C) in all the cells studied except in normal cells. However, combinations of CD45 inhibitors with IL-6 and/or vanadate with cytarabine worked antagonistically (B). Abbreviations: D, alendronate; A, allopurinol; B, balsalazide; C, cytarabine; JI, JAK inhibitor; IL-6, interleukin 6; V, vanadate. All experiments represent three independent biological replicates (n = 3). The error bar represents standard deviation of the mean from three repeats. Ex vivo experiments represent of peripheral blood cells of normal individuals (14 samples), AML (18 samples), and MPN (8 samples) patients

Fig. 6

Activation of apoptotic markers in response to combination of cytarabine with CD45 inhibitors. A Caspases 3 and 7 activities were significantly (p-value < 0.0001) increased by CD45 inhibitors (alendronate, D, allopurinol, A, and balsalazide, B), vanadate (V), JAK inhibitor (JI), and IL-6 combined with IC₅₀ cytarabine (C) expression on HEL cells, but not on UT-7 cells compared to DMSO control. B Expression of apoptotic markers including PARP and cleaved PARP, caspase 9 and 3, and cleaved 9 and 3. All experiments represent three independent biological replicates (n = 3). The error bar represents standard deviation of the mean from three repeats