c-Cbl is a RING finger based E3 ubiquitin ligase that is highly expressed in hematopoietic cells where it directs the polyubiquitylation and degradation of protein tyrosine kinases. c-Cbl also functions as a multi-domain adaptor that recruits and activates proteins such as PI 3-kinase. Oncogenic forms c-Cbl that have lost E3 ligase show enhanced activity of these adaptor functions in addition to the inability to downregulate activated tyrosine kinases.
c-Cbl mutations are found in patients with a range of myeloid malignancies that include myelodysplastic syndromes, chronic myelomonocytic leukaemia, juvenile myelomonocytic leukaemia, atypical chronic myeloid leukaemia and acute myeloid leukaemia. Sequencing has shown that the mutations are located in either the linker or RING finger domains and that these mutations abolish E3 ligase activity. Thus the development of leukemia is caused, at least in part, through the loss of this activity.
To investigate c-Cbl associated malignancies we generated a mouse with a knockin mutation in the RING finger domain that develops a myeloproliferative disease (MPD) progressing to lethal leukemia. The mutation is a substitution of a cysteine for alanine in the RING finger at 379 (i.e. C379A) that disrupts c-Cbl's ability to function as an E3 ubiquitin ligase. The majority of the c-Cbl C379A mutant mice succumb within a year with markedly elevated white blood cell (WBC) counts, splenomegaly and extensive infiltrations of myeloid cells into peripheral organs. These mice therefore provide a pre-clinical model for studying myeloid malignancies associated with oncogenic forms of c-Cbl.
We have found that hematopoietic progenitors from c-Cbl(C379A) mice have enhanced FLT3 signaling and an expanded population of FLT3high cells compared to WT and c-Cbl KO mice. When c-Cbl(C379A) mice are mated to FLT3 ligand KO mice the doubly mutant mice do not develop a severe MPD or leukemia and the FLT3high population is markedly suppressed. Thus targeting FLT3 may offer an effective therapy.
AC220 is a FLT3 inhibitor that shows excellent potency, selectivity and pharmacokinetic properties and is currently being investigated in clinical trials treating patients with activating FLT3 mutations. In this study we examined the effects of treating c-Cbl(C379A) mice with AC220 to determine whether AC220 can prevent the development of myeloid malignancies associated with aberrant wild-type FLT3 signaling.
To determine the effectiveness of AC220 for treating c-Cbl-associated MPD we established two cohorts, one comprising 16 C57BL/6.CD45.1 mice repopulated with bone marrow (BM) from an 8 week-old c-Cbl(C379A) mouse and one comprising 18 c-Cbl(C379A) mice aged 8–9 months. Mice were bled 5 days before dosing to determine pre-treatment WBC counts and both cohorts were then divided into two groups for daily dosing by oral gavage with either AC220 (10mg/kg) or vehicle (5% cyclodextrin) and bled at 3 and 6 weeks. Dosing with AC220 resulted in a marked decrease in WBC counts in all lineages. The mean WBC counts for the AC220-treated mice transplanted with c-Cbl(C379A) marrow at 3 and 6 weeks were 14.0 and 14.5×109/L respectively, compared to a mean WBC count of 33.5×109/L before treatment commenced. For the 8–9 month old c-Cbl(C379A) mice the 3 and 6 wk counts were 28.5 and 20.0 ×109/L respectively, compared to pre-treatment levels of 64.5×109/L. In contrast the WBC counts remained constantly high in the vehicle treated groups.
After 6 weeks of dosing the mice were sacrificed and the BM analyzed by flow cytometry to determine the effect on FLT3high progenitors. The AC220 treatment reduced the FLT3high population of LSK cells by more than two fold in both cohorts. Similarly AC220 treatment had a significant impact on spleen weights with a greater than two-fold reduction in the AC220-treated groups. In addition AC220 dosing resulted in a marked decrease in the myeloid invasiveness into peripheral organs, an outcome that suggests AC220 treatment would enhance survival.
These studies are the first to demonstrate in vivo that AC220 is a very effective treatment for diseases or conditions that are driven by WT FLT3. Future studies investigating aberrant FLT3 expression on progenitors from a range of MDS/MPN patients may therefore reveal therapeutic options that would not have otherwise been appreciated without the preclinical studies of the c-Cbl RING finger mutant mouse.
No relevant conflicts of interest to declare.
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