Introduction
Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder that occurs because of t(9;22)(q34;q11) translocations. CML prognoses markedly improved after the introduction of Abl tyrosine kinase inhibitors (TKIs). Since its approval in 2001 for frontline CML management, imatinib has proven to be effective in achieving high remission rates and improving prognosis. However, up to 33% of patients will not achieve an optimal response. Most patients with CML treated with imatinib will relapse if treatment is withdrawn, and numerous CML patients die due to
Abl mutation-related drug resistance and blast crisis. These circumstances have led researchers to develop a new generation of TKIs. Although second-generation TKIs, such as AMN107, appear to improve the treatment of CML, TKI resistance and relapse also frequently occur in patients.
de novo and secondary TKI resistance are significant problems for CML [
1‐
5]. Therefore, how to treat patients with CML who are resistant to Bcr-Abl tyrosine kinase inhibitors is an important and urgent issue for clinical hematology.
Moreover, TKIs have significant off-target inhibitory effects on multiple kinases. TKIs, through the off-target PPP2R5Cinhibition of kinases important for B-cell signaling, reduce memory B-cell frequency and induce significant impairment of B-cell responses in CML [
6]. TKIs also impair T cell function e.g., imatinib impairs CD8+ T cells specifically directed against leukemia-associated antigen function [
7].
Further advances in the treatment of CML may require the development of novel agents such as siRNAs that target specific CMLs or specific immunotherapies without significant toxicity that may have cooperative effects with TKIs [
8,
9]. siRNAs targeting the
Bcr-Abl and multidrug-resistance (
MDR-1) genes were used in an anti-CML study and demonstrated that a breakpoint-specific short-interfering RNA (siRNA) was capable of decreasing Bcr-Abl protein expression and antagonizing Bcr-Abl-induced biochemical activities [
10‐
12].
Synthetic small interfering RNAs (siRNAs) are promising gene-targeting agents that have shown great potential, particularly for development as specific anti-leukemia treatment [
13,
14]. A combination of
c-raf and
bcl-2 siRNAs induced apoptosis in HL-60, U937, and THP cell lines and increased chemosensitivity to etoposide and daunorubicin [
15].
Recently, we were the first to show that a higher
PPP2R5C expression level is found in peripheral blood mononuclear cells from chronic phase CML patients, and
PPP2R5C expression is significantly decreased in patients who achieved CR [
16].
PPP2R5C is a regulatory B subunit of protein phosphatase 2A (PP2A), which is one of the main serine-threonine phosphatases in mammalian cells, and it maintains cell homeostasis by counteracting most of the kinase-driven intracellular signaling pathways [
17]. The
PPP2R5C gene encodes five different spliced variants including B56γ1, B56γ2, B56γ3, B56γ5, B56γ6, and B56γ4, which is only found in mice. The locus for the functional
PPP2R5C gene is at 14q32.2, and a nonfunctional B56γ1 pseudogene for
PPP2R5C is located at 3p21.3 [
16‐
18].
PPP2R5C plays a crucial role in cell proliferation, differentiation, and transformation based on its induction of the dephosphorylation of p53 at various residues [
19]. It has been reported that the dynamic nuclear distribution of the B56γ3 regulatory subunit controls nuclear PP2A activity and may be responsible for the tumor-suppressive function of PP2A [
18]. Recently, alterations in the
PPP2R5C expression pattern that are associated with malignant transformation have been characterized in lung cancer, and the
PPP2R5C mutation F395C disrupts the B56γ–p53 interaction [
20].
To confirm the role of PPP2R5C in the proliferation of CML, we analyzed the effect of down-regulating PPP2R5C gene expression in imatinib-sensitive and imatinib-resistant chronic myeloid leukemia (CML) cell lines and primary cells from CML patients by RNA interference and confirmed the proliferation inhibition and apoptosis induction of PPP2R5C in CML cells.
Discussion
Targeted therapies are directed at unique molecular signatures of cancer cells to produce greater efficacy with less toxicity. The development and use of such therapeutics allow us to practice personalized medicine and improve cancer care [
27]. Imatinib is the first successful molecular drug specifically targeting the
Abl gene and has proven to be effective in achieving high remission rates and improving CML prognosis. Because TKI resistance and relapse frequently occur in patients, new targeted drugs that can specifically inhibit TKI-resistant CML urgently need to be developed. RNAi represents a new alternative for CML treatment that overcomes the difficulties of current drug treatments such as acquired resistance. The therapeutic targeting of
Bcr-Abl transcripts by siRNA was demonstrated in imatinib-resistant CML cells [
10,
28].
PPP2R5C plays a crucial role in cell proliferation, differentiation, and transformation based on its induction of the dephosphorylation of p53 at various residues [
19] and may be responsible for the tumor-suppressive function of PP2A [
18]. To confirm the role of PPP2R5C down-regulation on the inhibition of CML cells, particularly TKI-resistant CML cells, we used two
PPP2R5C siRNAs that target different exon sequences to analyze their effect on the inhibition of proliferation and apoptosis induction in CML cells. Moreover, to investigate the
PPP2R5C siRNA effects in imatinib-resistant CML cells, we selected two pairs of CML cell lines, including the imatinib-sensitive cell lines K562 and 32D-Bcr-Abl WT and the imatinib-resistant cell lines K565R, which lacks an
Abl mutation, and 32D-Bcr-Abl T315I, which has an T315I
Abl mutation, to compare the different changes induced by
PPP2R5C siRNA.
In general, RNAi effects are detected between 24 and 72 h after siRNA transfection. We demonstrated that the siRNAs effectively silenced PPP2R5C post-transcriptionally, and the control siRNA had no obvious influence 72 h after nucleofection. These results were confirmed at the RNA and protein levels. siRNAs targeting different exon domains had different efficacies for PPP2R5C gene silencing and subsequent biological consequences. Both PPP2R5C siRNAs demonstrated significant effects on the knockdown of PPP2R5C expression in CML cell lines, and PPP2R5C-siRNA799, which targets exon six, demonstrated robust knockdown of PPP2R5C expression in K562R cells at all time points.
There are reports that siRNAs targeting
Bcr-abl increased sensitivity to imatinib in Bcr-Abl-overexpressing cells and cells expressing the imatinib-resistant Bcr-Abl kinase domain mutations H396P and Y253F [
10,
28]. There are no reports regarding the effects of the suppression of
PPP2R5C on changing cell biological functions. Our previous study first demonstrated that the suppression of
PPP2R5C by RNAi effectively inhibited the proliferation of the Molt-4 and Jurkat cell lines; however, the suppression of
PPP2R5C by RNAi could not significantly induce apoptosis in Molt-4 and Jurkat T cells [
22]. In contrast, the
PPP2R5C siRNAs not only inhibited cell proliferation but also induced apoptosis in imatinib-sensitive and imatinib-resistant CML cell lines. These results indicated that down-regulating PPP2R5C could significantly inhibit the proliferation of CML cells, and the underlying mechanism might be different between CML and T-ALL cells. More importantly, we found a significantly higher inhibition effect in K562R cells treated with
PPP2R5C-siRNA799, and the inhibition effect in 32D-Bcr-Abl T315I cells, which have a T315I
Abl mutation, was similar to that of 32D-Bcr-Abl WT cells. Such effects are particularly important for the targeted therapy of imatinib-resistant CML cells that either lack an
Abl mutation and have primary and imatinib-induced resistance or those with an
Abl T315I mutation, which resist new-generation TKIs. Therefore, it is interesting to analyze the molecular mechanism of
PPP2R5C siRNA-mediated cell proliferation suppression in different leukemia cells. It has been reported that TKI-resistant, Philadelphia chromosome-positive cell lines without an
Abl mutation are unique because they dephosphorylate ERK1/2 and STAT5 after imatinib treatment, while PI3K/AKT1/mTOR activity remains unaffected. The inhibition of AKT1 leads to apoptosis in imatinib-resistant cell lines. Therefore, these Ph + cell lines show a form of imatinib-resistance attributable to the constitutive activation of the PI3K/AKT1 pathway [
3]. Whether down-regulating PPP2R5C contributes similar effects to the inhibition of PI3K/AKT1/mTOR signaling requires further investigation. We also found that
PPP2R5C siRNA could inhibit the proliferation of primary CML cells in limited experiments, and this effect should be further explored using a larger patient cohort.
Moreover, whether
PPP2R5C siRNAs could potentiate the efficacy of TKIs in imatinib-resistant cells is worthy of further investigation. One study has shown a synergistic effect between AMN107 and arsenic trioxide (ATO) or
Bcr-Abl-siRNA in the K562R imatinib-resistant cells or those with an H396P abl mutation, indicating that the combination of AMN107 and ATO or siRNA may represent a new strategy for the treatment of imatinib-resistant CML patients [
10,
29].
In conclusion, our findings provide evidence for the effect of proliferation inhibition and apoptosis induction in CML cells by PPP2R5C knockdown, and such effects may particularly benefit developing a strategy including a combination of targeted therapy using TKIs for resistant cells. A successful clinical trial demonstrated that the in vivo application of targeted nonvirally delivered synthetic
Bcr-abl siRNA in a female patient with recurrent CML that was imatinib resistant (Y253F mutation) and chemotherapy after an allogeneic hematopoietic stem cell transplantation could silence the expression of the
Bcr-Abl gene [
28]. These data imply that siRNA may be suitable for development as a specific anti-leukemia treatment. However, a recent study demonstrated that the phosphatase activity of PP2A is suppressed in chronic myeloid leukemia and other malignancies characterized by aberrant oncogenic kinase activity, and preclinical studies show that the pharmacological restoration of PP2A tumor-suppressor activity by PP2A-activating drugs (e.g., FTY720) effectively antagonizes cancer development and progression [
30]. These findings appear to be contrary to our results, and further characterization of the function of the different regulatory B subunits of PP2A and a discussion of the different effects on the different PP2A target subunits is needed.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YQL contributed to concept development and study design. QS performed the nucleofection, real-time PCR, cell proliferation assays and apoptosis analysis, SCL performed the immunoblotting and apoptosis analysis, YC screened the highly efficient and specific PPP2R5C siRNAs, and LJY, SHC, XLW and BL helped to collect samples and perform cell culture. YHL and KEZ was responsible for the collection of clinical data. YQL and QS coordinated the study and helped draft the manuscript. All authors read and approved the final manuscript.