Sensitivity of a Homozygous c-Cbl Mutant AML Cell Line (JPM11) to Tyrosine Kinase Inhibitors, Implications for Potential Targeted Molecular Therapy.
Authors: Makishima Hideki , Sugimoto Yuka , Szpurka Hadrian , Prince Courtney , Clemente Michael , Muramatsu Hideki , Schade Andrew E. , Maciejewski Jaroslaw P. 
Published: October 2019

Abstract
Abstract 2607
Poster Board II-583
Acquired somatic copy-neutral loss of heterozygozity (CN-LOH) also referred as to uniparental disomy (UPD) has been frequently found in MDS, MDS/MPN or secondary AML and may point towards genes harboring homozygous mutations. UPD11q23.3 is present in 7% of patients with CMML and 11% of AML derived from this condition. This recurrent UPD has been shown to coincide with homozygous c-Cbl mutations mostly located in ring finger or linker domain (RFD) of the gene. c-Cbl is a member of the E3 ubiquitin ligase Cbl family, which poly- or monoubiquitinate activated Src family kinases (SFK) and various receptor tyrosine kinases (RTK). Inactivation of ubiquitination activity through mutations of RFD may lead to enhanced and/or prolonged receptor signaling, a function which can contribute to the clinical phenotype of patients with c-Cbl mutation. In contrast to patients with UPD11q, those LOH due to del11q were only rarely affected by hemizygous c-Cbl mutations and only 4/442 (0.9%) with heterozygous mutation of c-Cbl were identified. c-Cbl mutant cases are associated with monocytosis, monocytoid blasts and aberrant c-kit expression. Serial studies showed acquisition of c-Cbl mutations during malignant evolution. Our analysis of treatment outcomes for 17 c-Cbl and Cbl-b mutant vs. 307 WT cases (MDS, MDS/MPN, MPN and AML), showed that patients with Cbl mutations were treated with intense chemotherapy or stem cell transplantation, suggesting that these therapies were more frequently selected due to the aggressive biology of the Cbl mutation-associated disease. When we performed univariate analysis of survival impact of various clinical parameters in 284 screened cases, significant differences in WBC, monocyte counts, disease risk and the presence of Cbl family gene mutations were found. In multivariate analyses, Cbl mutations (hazard ratio 2.2 (95%CI; 1.2-4)) (p=.013) were shown to be an independent adverse factor for overall survival, as well as advanced stage of diseases. Poor prognosis associated with Cbl mutations necessitates new treatment approaches and identification of pathogenic pathways resulting from block of ubiquitination activity of c-Cbl may provide clues as to possible molecular targets. SNP-array screening of 33 myeloid leukemic cell lines revealed 3 with LOH11q. By sequencing, we identified a homozygous R420Q missense mutation in a cell line (JPM11) with UPT11q and it was derived from CMML transformed to sAML. This RFD mutation affects ubiquitination activity and was also reported to be associated with cytokine-independent growth. We found the mutation in the same position in 4 clinical cases (19% of all c-Cbl mutant cases; 503 of patients screened). We then used JPM11 cell line as a model to study agents with potential activity in c-Cbl mutant leukemias. Forced expression of WT c-Cbl cDNA prevented growth of JPM11 suggestive of the dependence on the absence of WT c-Cbl and consistent with homozygozity for c-Cbl mutation found in patients. In contrast, partial shRNA knockdown of the mutant expression resulted in a marginal decrease in the proliferative capacity. There were no effects of c-Cbl overexpression or partial knockdown observed on cell lines with WT c-Cbl. We have compared the differential effects of various RTK and SFK inhibitors, including sunitinib, sorafenib, dasatinib, and PP2 on proliferation of JPM11 with 4 WT c-Cbl cell lines. In addition, to investigate other pathways potentially affected by RFD knockout, and located likely downstream of RTK and SFK, we have also studied the potential role of mTOR, as well as PI3K and MAPK. The selectivity of inhibitory effects was calculated by as a ratio between mutant/WT cell lines. JPM11 showed increased sensitivity to SFK inhibitors; dasatinib (LD50 50nM vs. >1000nM) and PP2 (LD50 10μM vs. >20 μM). Multi-RTK inhibitors demonstrated almost equal responses between JPM11 and WT cell lines and blocked growth of all cell lines irrespective of the c-Cbl function at 2.5–10μM (sunitinib) and 5–20μM (sorafenib). In contrast, inhibitors against mTOR (rapamycin), PI3K (LY294002) or MAPK (UO126) were ineffective in comparable studies. Based on the inhibition spectrum, our results indicate that activating multiple pathways downstream of RTK or SFK are involved in the pathogenic process mediated by c-Cbl mutation while downregulation of any single cascade including mTOR, PI3K and MAPK pathway may not be enough for the reduction of pro-oncogenic effect of mutant c-Cbl.

Disclosures:
No relevant conflicts of interest to declare.