Prohibitin Expression Level As a Companion Diagnostic Marker in Cytogenetically Normal Acute Myeloid Leukemia

Background: Cytogenetically normal acute myeloid leukemia (CN-AML) accounts for almost half of all AML and shows significantly heterogeneous clinical outcomes. Despite the advancements in the molecular pathophysiology of CN-AML, clinical outcome of this leukemia remains unsatisfactory. The recent studies of close relationship between mitochondrial alterations and pathologies of various tumors and degenerative diseases suggested that mitochondria have been an important target for the identification and development of novel companion diagnostic and prognostic marker. These findings prompted us to investigate whether mitochondrial markers could serve as a potential diagnostic, prognostic and therapeutic target in CN-AML patients.

Patients and Methods: The proteome analysis of the mitochondria enriched cytoplasmic fractions in primary CN-AML and counterpart normal cells was performed using 2-DE and MALDI-TOF/TOF mass spectrometry. For the development of anti-leukemic drug candidates targeting PHB in CN-AML cells, we synthesized potent chemical substances that can alkylate identified biomarkers. Quantitative reverse transcriptase-polymerase chain reaction and immunohistochemical staining (IHCS) on paraffin-embedded bone marrow sections were performed in order to obtain clinicopathological implications of candidate biomarkers. To assess the functional significance of identified biomarkers, we investigated the effect of β-catenin accumulation on the expression of identified biomarkers because accumulation of β-catenin in the nucleus has been identified in leukemic cells.

Results: Prohibitin ( PHB /gi4505773) was highly expressed in primary CN-AML cells compared with normal cells. PHB2 protein expression using IHCS was assessed in 133 CN-AML patients. Patients with PHB2 overexpression showed inferior OS (23.7% vs 50.0%; P =0.026) and high hazard ratio (1.808; P = 0.028) compared to PHB2 expression negative patients in CN-AML. Quantitative expression of PHB1 g ene (ddC_t) was higher in the control group; 0.31 to 28.32 (mean 4.40, median 2.16) in control group versus 0.22 to 6.52 (mean 1.83, median 1.56) in patient group. Meanwhile, PHB 2 gene expression was higher in the patient group; 0.0 to 0.46 (mean 0.16, median 0.15) in control group versus 0.15 to 3.49 (mean 0.80, median 3.49) in patient group. As a result of linear discrimination analysis, a linear equation (y = 0.5857 * x₁ - 4.7252 * x₂ - 0.18652; x₁ was ddC_t of PHB1 , x₂ was ddC_t of PHB 2) was derived. Estimating the prevalence of AML as 0.003%, the sensitivity of newly developed algorithm was 88.0%, specificity was 87.0%, negative predictive value (NPV) was 99.9% and positive predictive value (PPV) was 2.0%. The elevated level of PHB in CN-AML cells was the result of Wnt signals that were up-regulated in AML cells. PHB is a direct target of β-catenin, and TCF-4/LEF-1 binding motif, CATCTG, appears in the promoter region of PHB by site-directed mutagenesis and ChIP assay. This β-catenin-mediated activation of PHB expression is independent of c-MYC activation which is also a product of Wnt signaling. Potent chemical substances that can alkylate PHB in AML cells, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU). Proliferation suppression was observed in leukemic cell lines, THP-1, K-562 and Kasumi-1, which were treated with CCEU and ICEU in time and dose dependent manner. Moreover, notable morphological transformation of leukemic cells was observed when treated with 50 - 200 umol of CCEU and ICEU for 24 hours. Cell cycle analysis of CCEU-and-ICEU-treated- THP-1 and Kasumi-1 cell lines showed a remarkable increase of the sub-G1 phase.

Conclusions: Overexpression of PHB2 protein was associated with poor prognosis in CN-AML. Newly developed algorithm using quantitative data of PHB1 and PHB2 mRNA expressions also showed the possibility as rapid diagnostic tools for leukemic cell identification, and novel alkylating chemotherapeutics, CCEU and ICEU, targeting PHB for selective killing of leukemic cells have been developed. PHB2 expression status can provide clinically valuable results for the diagnosis, prediction of the prognosis, detection of minimal residual disease and therapeutic targeting of AML with normal karyotype. High expression of PHB was served as a companion diagnostic marker in cytogenetically normal acute myeloid leukemia.