PKC-Epsilon Overexpression Is Associated with Poor Outcomes in AML and Promotes Chemoresistance and Hematopoietic Stem Cell Quiescence

Acute myeloid leukemia (AML) is a heterogeneous group of hematological malignancies which are characterized by the accumulation of clonal, abnormally differentiated blasts in the bone marrow. For most patients, prognosis remains poor as a large proportion relapse following a period of complete remission.

Protein kinase c epsilon (PKCε) is a pleiotropic regulator of cell proliferation, differentiation, apoptotic and survival signaling. Uniquely in the PKC family, PKCε also has demonstrated oncogenic properties and high expression of PKCε has been associated with poor patient outcomes, disease aggressiveness, and chemoresistance in solid cancers. A role for PKCε has also been implicated in AML; however, its clinical significance has not been elucidated.

To address this, we first determined whether PKCε was aberrantly expressed in AML. Patient samples were collected from the MRC AML14 and AML15 clinical trials and quantitative estimation of PKCε at the protein and mRNA level was conducted. PKCε overexpression (>2SD of mean of controls) occurred in 32% (43/132) of patients based on mRNA expression and 37% (26/70) of samples based on protein expression, compared to normal human CD34⁺ cells. In addition, a positive correlation between PKCε mRNA and protein expression was established (R=0.64, p<0.01). We next evaluated the clinical impact of high PKCε expression in AML using the TCGA RNAseq dataset. High PKCε expression (>median), correlated with significantly worse overall survival (95% CI (34-54); p<0.05), with the median survival for being 11 months, compared with 24 months for patients with low PKCε expression. High PKCε expression did not correlate with age, molecular abnormality, gender, or WBC, and is therefore likely to be an independent prognostic factor.

To study the mechanisms through which PKCε could contribute to poor outcomes in AML, the influence of PKCε overexpression on the chemosensitivity of AML cell lines was explored. In the AML cell lines investigated (U937, HEL, TF-1 and NOMO-1), PKCε overexpression was associated with significantly increased daunorubicin (DNR) resistance in U937 and HEL cells (2.6-fold, n=4; p<0.05 for both lines) without affecting their sensitivity to cytarabine, suggesting that PKCε can selectively promote resistance to DNR. The therapeutic mechanism of DNR is, in part, accounted for by the generation of reactive oxygen species (ROS) and we are currently investigating whether PKCε expression protects against ROS-generating agents as has previously been suggested.

We next examined the effect of PKCε on normal human hematopoiesis. RNAseq data from both human and murine hematopoietic cells show that PKCε is endogenously expressed in normal hematopoietic cells, with the highest levels of expression occurring in the HSC compartment and monocytes. Overexpression of PKCε in human CD34⁺ progenitor cells promoted myeloid differentiation of unilineage monocyte and granulocyte progenitors which showed an increase in expression CD14 and CD15, with CD11b being significantly upregulated on monocytes and granulocytes by 1.4-fold±0.3 (n=4, p<0.05) and 1.6-fold±0.2 (n=3, p<0.05), respectively. Conversely, shRNA mediated PKCε knockdown in these cells reduced the expression of these myeloid markers, supporting a role for PKCε in developmental regulation of the myeloid lineage.

We next examined the effect of PKCε overexpression on the self-renewal and hematopoietic activity of human HSC (CD34⁺CD38^-). Transduced HSC were scored for colony formation, in addition to being seeded onto MS5 stroma and assessed for their capacity to generate cobblestone area forming cells (CAFC). PKCε had no significant impact on colony forming efficiency but ablated CAFC formation suggesting that PKCε selectively promotes quiescence of HSC (n=2). Since quiescence of leukemic stem cells (LSC) is widely thought to contribute to relapse in AML, this may represent a further mechanism through which PKCε expression confers a poor outcome in this disease and we are currently investigating this.

In summary, we show that elevated expression of PKCε occurs frequently in AML and our data indicate it is an independent prognostic marker of poor outcome in this disease. Experimental models suggest that overexpression of PKCε may contribute to treatment resistance through promoting DNR resistance and potentially by promoting LSC quiescence.