Targeting CDK6 and BCL2 Exploits the “MYB Addiction” of Ph+ Acute Lymphoblastic Leukemia

Background. Philadelphia-Chromosome positive acute lymphoblastic leukemia (Ph+ ALL) is an aggressive disease with a 5-year survival rate of less than 50%. BCR-ABL1 tyrosine kinase inhibitors (TKI) in combination with chemotherapy typically fail to induce long-lasting remission in part due to BCR-ABL1 dependent TKI resistance and in part because of BCR-ABL1 independent mechanisms. These findings suggest that the identification of additional targets is necessary for a more effective management of the disease.

Results. We showed previously that Ph+ ALL cells and other types of leukemia are particularly reliant on MYB expression for their growth and survival. To support the essential role of MYB in Ph+ ALL we assessed the biological effects induced by MYB silencing in Ph+ ALL cell lines (BV173 and SUP-B15). In our studies we found that: i) MYB silenced Ph+ ALL cells display reduced growth and colony formation in methylcellulose due to a block in the G1-phase of the cell cycle; ii) such an effect was followed by increased apoptosis detected by Annexin-V staining and caspase-3 cleavage. Moreover, doxycycline-induced MYB silencing in NOD/SCID-IL-2Rγnull (NSG) mice injected with Ph+ ALL cells markedly suppressed leukemia development. These results suggest that MYB would be an ideal candidate for targeted therapies; however, no direct inhibitor for MYB is currently available and little is known about MYB function in primary Ph+ ALL cells. By performing transcriptome analysis of MYB silenced Ph+ ALL cell lines we identified several cell cycle regulatory genes as transcriptional targets of MYB including CDK6, CCND3 and CDKN1A . In particular, CDK6 is critically important for MYB dependent regulation of cell cycle progression because CDK6 silencing induces the cell cycle arrest of Ph+ ALL cells. By contrast, expression of CDK4 was not regulated by MYB and was not required for the proliferation of Ph+ ALL cells, a finding likely explained by the fact that by immunofluorescence, CDK6 is readily detected in the nucleus of Ph+ ALL cells, whereas CDK4 appears exclusively in the cytoplasm. MYB silencing led to a marked decrease in BCL2 expression, concomitantly with the induction of apoptosis. Ectopic expression of BCL2 and CDK6 rescued the growth and survival of Ph+ ALL cells in vitro ; however, it restored in vivo leukemia formation only in part, suggesting that expression of additional MYB targets is required to re-establish the full leukemogenic potential of MYB-silenced cells. We observed that Ph+ ALL cells are remarkably sensitive to CDK4/CDK6 inhibition by Palbociclib confirming the important role of CDK6. On the other hand, the selective BCL2 inhibitor Venetoclax displayed sample-to-sample variation in inducing apoptosis of Ph+ ALL cell lines and primary samples, probably due to variable expression of additional members of the BCL2 family. To overcome this limitation, we examined the anti-leukemia effects of the pan-BCL2 inhibitor Sabutoclax in combination with Palbociclib. The Palbociclib-Sabutoclax combined treatment synergistically reduced the viability of BV173 cells. In addition, NSG mice injected with blast cells from two Ph+ ALL patients were treated for ten days with Palbociclib 150 mg/kg and/or Sabutoclax 5 mg/kg (every other day) and the leukemia burden was analyzed in the peripheral blood before and at bi-weekly intervals after the treatment. By comparing the percentage of circulating leukemic cells before and after the treatment, Palbociclib suppressed Ph+ ALL leukemia development and caused a moderate reduction in the leukemia burden (17% and 32% decrease) while Sabutoclax had negligible effects. However, the Palbociclib-Sabutoclax combination induced a much stronger decrease (77% and 90%), significantly superior to that induced by each drug alone.

Conclusions. These results indicate that the oncogenic effects of MYB in Ph+ ALL is primarily due to the transcriptional activation of CDK6 and BCL2. Furthermore, our xenograft studies constitute a proof-of-principle that the pharmacological targeting of CDK6 and BCL2 is an effective strategy to exploit the MYB "addiction" of Ph+ ALL cells.