JAK2-Mediated Extrinsic Survival of CML Stem Cells: Exploring the Potential Combination of BCR-ABL and JAK2 Inhibitors In Vivo

The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib are very effective for the treatment of chronic phase CML. However, the majority of these patients continue to have persistence of CML cells despite continued therapy, suggesting that TKIs fail to target leukemic stem cells (LSCs). There is increasing evidence that the bone marrow microenvironment provides a sanctuary to LSCs, thereby contributing to persistence.

We used the human stromal cell lines (HS-5, HS-23, HS-27a) to model the microenvironment. Conditioned media from HS-5, but not HS-23 or HS-27a, reduced apoptosis of CML cell lines treated with TKIs (K562, LAMA-84, KBM-5 and KYO-1), consistent with previous reports. Similarly, CML CD34+ cells were protected from 5 μ M imatinib in a 4 day co-culture with HS-5 cells, as assessed by CFU-GM colony survival (23% vs 9% when compared to untreated controls, N=6, p=0.018). We were also able to demonstrate protection from TKIs with transwells over HS-5 and with HS-5 conditioned media, which suggests that factors secreted by HS-5 cells protect CML cells from TKIs. Cytokine analysis of conditioned media revealed relatively higher concentrations of IL6, IL-8, MCP-1, MCP-3, G-CSF and GM-CSF from HS-5 as compared to HS-23 and HS-27a. Since IL-6, G-CSF and GM-CSF are known to signal via JAK2, we tested combinations of imatinib and JAK2 inhibitors (TG101209 or CYT387) using our in vitro assay. Combination treatment with imatinib and CYT387 or TG101209 abrogated the protective effects of HS-5 conditioned media in CML cell lines. Combination treatment of CML CD34+ in HS-5 co-culture assays also abrogated the protective effects of stroma on colony formation. However, we observed that both normal CD34+ and CML CD34+ colony formation was dramatically reduced by JAK2 inhibitors using our HS-5 co-culture system, particularly at higher doses. Thus, it was unclear if a potential therapeutic window existed in vivo.

To test the potential of combination therapy in vivo, we infected marrow from Balb/c mice with a retrovirus that simultaneously expresses BCR-ABL and GFP, followed by transplantation into lethally irradiated syngeneic recipients. The mice were separated into five cohorts: vehicle control, TG101209 (200mg/kg/d), nilotinib (75mg/kg/d), nilotinib + low-dose TG101209 (50mg/kg/d) and nilotinib + high-dose TG101209 (200mg/kg/d). The vehicle-treated control group died rapidly of myeloproliferative disease (MPD) with a median survival of 15.5 days. The median survival of mice treated with TG101209 was slightly prolonged at 20.5 days (p=0.06); however, these mice also died of MPD with enlarged spleens/livers and lung hemorrhage. The survival curves of mice treated with nilotinib monotherapy and nilotinib + low-dose TG101209 were similar (median survival not reached at termination of experiment). Mice treated with nilotinib + high-dose TG101209 initially had minimal mortality, however on day 26 the mice began to die without signs of MPD (no definitive cause of death at autopsy) and thus the remaining cohort was sacrificed on day 27 for analysis. In stark contrast to the other cohorts, the spleens of these mice were very small, leukopenic, and largely devoid of normal follicles, with decreased spleen weight compared to mice treated with nilotinib monotherapy (0.025 vs. 0.072 gm, p<0.01). The bone marrow was also profoundly hypocellular, suggesting that myelosuppresion (anemia and leukopenia in particular) may have been a factor in mortality. However, despite toxicity this combination may retain a degree of selectivity for BCR-ABL cells since disease burden, as measured by the percentage of GFP-positive cells, was reduced compared to nilotinib monotherapy (spleen: 4% vs 11.7%, p=0.047; bone marrow: 8.7% vs 13.8%, p=0.22).

(1) Factors secreted by human bone marrow stromal cells attenuate the effects of imatinib in CML cell lines and primary CML CD34+ cells in a JAK2-dependent fashion. (2) Simultaneous in vivo inhibition of BCR-ABL and JAK2 dramatically reduces BCR-ABL expressing cells, but at the cost of marrow toxicity. We speculate that this limitation may be overcome by intermittent rather than continuous JAK2 inhibition, a strategy that might avoid toxicity while reducing persistent BCR-ABL disease burden.

Deininger:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Genzyme: Research Funding; Ariad: Consultancy.