Proteomic Profiling of 112 Proteins in Chronic Myeloid Leukemia Patient Samples Using Reverse Phase Proteins Arrays (RPPA) Reveals Distinct Protein Expression Signatures Associated with Advanced Phase Disease and the CD34+ Compartment

Having previously shown that protein expression signatures, based on the activation state of cell cycle, apoptosis and signal transduction regulating proteins, existed and were prognostic in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), we extended this to evaluate protein expression patterns in CML.

We have generated RPPA using protein derived from the leukemia-enriched fraction of 40 primary CML samples with the goal of defining comprehensive proteomic expression patterns in CML. Of the 40 patient samples included in this analysis, 25 were in chronic (CP), 5 in accelerated (AP), and 10 in blast phase (BP). Of the latter, 6 were lymphoid BP and 4 myeloid BP. All protein preps were made from fresh cells on the day of collection. Present as controls were 16 CD34+ BM and 9 normal PB lymphocyte samples. Samples are printed as 5 serial 1:2 dilutions in duplicate using an Aushon 2470 Arrayer. Each array has a total of 6912 dots printed. Slides were probed with 112 antibodies (ABs) against apoptosis, cell cycle, signaling (STP), regulating proteins, integrins, and phosphatases among other functional protein groups, including 85 vs. total protein, 22 vs. phospho-specific sites and 5 vs. caspase or PARP cleavage sites. Spot intensities were quantified using MicroVigene software. Data was analysed using R, with loading control and topographical background normalization being utilized.

We first tested the differences of the protein expression between patients with CP, AP, and BP. To that end, we centered proteins on the median across all samples. An ANOVA analysis revealed 20 proteins (from a total of 112 proteins probed) were differentially expressed across the different phases of CML by using a minimum statistical significance cutoff of p<0.01. The expression of proteins such as HSP90, RB, AIF, PP2A, BCL2, XIAP, SMAD1, SSBP2α, PARP, GAB2, and TRIM24 was low in patients with CML-CP but progressively increased as patients progressed to BP, with samples obtained from patients with CML-AP exhibiting intermediate levels between CP and BP. Conversely, the expression of PKCΔ.p664, AKTpT308, actin, p70S6Kp, Rac1.2.3, PDK1p, MEK, and CDK4 decreased gradually as patients progressed from CP to BP, with samples obtained from patients with CML-AP exhibiting intermediate levels. Notably, downregulation of genes involved in the Ras-MEK-MAPK pathway and upregulation of those encoding cytoskeletal and adhesion proteins (actin, Rac) has been previously reported in gene expression profiles (Radich et al, PNAS 2006).

A similar analysis was conducted to investigate differences in protein expression between the CD34+ (23 samples) and CD34- (37 samples) compartments, the former of them putatively containing the CML stem cell population. A t test was used for each protein and those whose expression were significantly (p<0.01) differentially expressed were selected. Forty-two proteins were identified as differentially expressed in CD34+ CML cells, including upregulation in the CD34+ compartment of those involved in the WNT/β-catenin (TCF4, survivin), adhesion proteins (integrin-β3, FAK, SRC), STAT pathway (STAT3, STAT3p705, Bcl_XL), PARP, pPTEN, MYC, pPKCα, mTOR, and PP2A. Conversely, several proteins were downregulated in CD34+ cells: Mdm2, p38, MEK, AKTpT308, NFκB pathway (PKCΔ, NFκB.p65, SHIP1), and proapoptotic proteins (BID, BIM). When segregated according to BCR-ABL1 mutational status, no differences in protein expression were observed between samples carrying the F317L or the T315I mutations versus all other mutations. Similarly, no differences in protein expression were observed between patients carrying unmutated BCR-ABL1 and those carrying a mutant BCR-ABL1 enzyme.

We have identified by RPPA technology specific subsets of proteins whose expression is associated with CML progression. Likewise, specific proteins appear to be differentially expressed in the CML stem cell compartment. These proteins might represent therapeutic targets.

No relevant conflicts of interest to declare.