Resistance to imatinib (IM) has posed a great obstacle in treating Chronic Myeloid Leukemia(CML). New generations of tyrosine kinase inhibitors (TKIs) have been developed for BCR-ABL dependent IM resistance, which is commonly due to BCR-ABL kinase-domain mutations. However, in 50% or more of IM-resistant CML patients there is no mutation in BCR-ABL, and intrinsic resistance of CML stem cells often causes disease relapse. Understanding the mechanism of BCR-ABL independent IM resistance is therefore essential for devising strategies to eradicate resistant and residual leukemia.
Through a genome-wide shRNA screen in a IM-sensitive human CML cell line, K562 cells, we identified 16 imatinib-sensitizing genes (IMSGs), knockdown of which increased IM resistance of multiple human CML cell lines (e.g., K562, KYO-1 cells. >2 folds increment), and primary CML mice bone marrow cells (> 2 folds of CFUs). Knocking down 11 IMSGs increased IC50IM by more than 5 folds, and also conferred equivalent level of resistance to the second generation TKI, Dasatinib. Extensive signaling pathway analysis by immune-blotting for characteristic protein phosphorylation markers revealed co-activation of PKC and MEK/ERK pathway in majority of IMSG knockdown K562 cell lines. qRT-PCR analysis in IMSG knockdown cells identified PRKCH as the predominant deregulated PKC family member. Mild overexpression of PRKCH increased IC50IM by 10-20 folds, and in vitro phosphorylation confirmed that PRKCH could directly phosphorylate and activate CRAF, therefore MEK/ERK pathway. PRKCH is highly expressed in IM-resistant samples with wildtype BCR-ABL compared to those with mutant BCR-ABL (p<0.05, Mann Whitney test) or IM-sensitive samples (p<0.01, Mann Whitney test). Accordingly, multiple IMSGs were also significantly down-regulated (i.e.,CLEC5A, ELF5, WNT7B, p<0.05), supporting a suppressive role of diverse IMSGs on PRKCH expression. Specifically, we found ELF5 acted as a transcription repressor of PRKCH.
To investigate whether simultaneous inhibition of BCR-ABL and MEK/ERK signaling could more efficiently kill BCR-ABL-independent IM-resistant CML cells, we analyzed the effect of combining IM treatment with a FDA-approved MEK inhibitor, Trametinib, in multiple BCR-ABL independent IM-resistant CML mouse models, and primary resistant CML cells. Treatment with both drugs showed a substantially greater effect than either drug alone, and in many instances, the effect of combined drug treatment was synergistic.
qRT-PCR analysis showed that PRKCH expression in IM-resistant CML stem cells is higher than that in IM-sensitive CML progenitors (4~25 folds, p<0.01, Student’s t test), implying an important role of PRKCH in CML stem cell resistance. We established a PRKCH knockdown CML mouse model by co-expressing BCR-ABL and a PRKCH shRNA and performed stem cells analysis with or without IM treatment. Although both PRKCH knockdown and IM treatment abolishes MEK/ERK signaling in CML progenitors, only PRKCH controls MEK/ERK pathway in CML stem cells while IM barely has any effect. Combined IM treatment with PRKCH knockdown or MEK inhibition substantially increased the apoptosis rate of CML stem cells (p<0.01, Student’s t test), and prolonged CML mice survival(n=5, p<0.05, Log-rank test).
In addition to MEK/ERK pathway, we found PRKCH could activate PKD2, a downstream effector of novel PKCs. PRKCH expression increases upon IM treatment, along with increased PKD2 activity (S916 phosphorylation) in multiple CML cell lines(e.g., K562, KYO-1, LAMA84). PKD2 expression was also induced by IM in primary human CML stem cells. PKD2 promotes cell survival by activating NF-kB pathway, and IM treatment increased p65/RelA phosphorylation at S536 and its nuclear translocation, together with over 4 folds increase in expression of anti-apoptotic p65 target genes (e.g., CIAP2, Bfl-1/A1). Pharmacological inhibition using pan-PKD inhibitor, CRT0066101, completely abolishes IM induced p65 phosphorylation and increased the killing of CML cells by IM.
This study for the first time reveals the critical role of PKC pathway in BCR-ABL independent IM resistance in CML. The validation of MEK/ERK and PKD/NF-kB pathway as critical downstream effectors provided specific targets for small molecule therapy in TKI-resistant CML and other malignancies caused by deregulated PKC pathways.
Cerny:Cellerant Therapeutics: Honoraria, Research Funding.
Asterisk with author names denotes non-ASH members.