Dual Targeting of Ph+ ALL with Dasatinib and ABT-199 (Venetoclax)

Introduction:

Treatment of adult Philadelphia chromosome positive acute lymphoblastic leukemia (Ph⁺ALL) remains a challenge. While the addition of the targeted tyrosine kinase inhibitors (TKI) to standard cytotoxic therapy has greatly improved upfront treatment, treatment related mortality in older adults remains high. A novel induction regimen combines the targeted dual Abl/Src TKI Dasatinib (Sprycel, BMS) with a corticosteroid. After the first 21 days of induction the corticosteroids are tapered due to significant toxicities, particularly in older adults. Unfortunately, remaining on TKI monotherapy renders patients susceptible to the development of TKI resistance and thus identifying targeted agents that could enhance the activity of TKIs is urgently needed.  Recently a novel and selective inhibitor of BCL-2, ABT-199 (Venetoclax, AbbVie) has shown impressive activity against other lymphoid malignancies including CLL and NHL.    Here we describe the pre-clinical and in vivo efficacy of ABT-199 in combination with dasatinib in Ph⁺ALL and propose its potential use in future clinical trials.

Methods:

Drug efficacy in vitro was determined using the Ph⁺ALL cell line SupB15, primary Ph⁺ALL sample (12-149), the dasatinib sensitive Pre-B ALL cell line RCH and the CML cell line K562.  Cells were treated with dasatinib, ABT199 or in combination for 72 hours.  Cell viability was assessed with the colorimetric MTS assay and apoptosis was assessed with annexin V staining. Expression of the BCL family proteins BCL-2 and MCL-1 were assessed via immunoblot.  Immunodeficient NSG mice were injected with 12-149, then one week later treated with vehicle, 5 mg/kg dasatinib, 5 mg/kg ABT-199, or the combination daily for 5 days each week.   Peripheral blood was obtained every 1-2 weeks to assess for engraftment as defined by the presence of >10% human CD45+ cells in the peripheral blood.  Once engrafted, mice were euthanized and examined.  Mononuclear cells were extracted and assessed for BCL2 and MCL1 expression.  Statistical methods were performed using Calcusyn and PRISM.

Results:

Susceptibility to BCL2 inhibition:  Of the dasatinib sensitive cells tested, SupB15 and 12-149 cells were susceptible to ABT-199 while RCH and K562 cells were not.  The ALL cells expressed BCL-2 while the CML cell line expressed BCLx.  SupB15 expressed low levels of the antiapoptotic protein MCL1 while RCH cells had relatively higher levels.  siRNA of MCL-1 rendered the RCH cells sensitive to inhibition by ABT-199.  In SupB15 cells, treatment with ABT-199 alone led to upregulation of MCL-1 at 24h which was prevented by the combination of dasatinib + ABT199. Synergy in Ph⁺ALL: The calculated IC₅₀ of dasatinib and ABT199 in SupB15 were 8.8nM and 5.9nM, respectively. The IC₅₀ of equimolar combination was 0.42nM, and synergistic with combination index (CI) values between 0.15 and 0.49.   Primary Ph⁺ALL xenograft cells showed a similar pattern of synergy to the dasatinib + ABT199 combination.   Combination treatment also greatly increased apoptosis as measured by Annexin V staining.  Xenograft Studies: Animals were treated with a ten-fold lower dose of dasatinib and ABT199 from prior published data.  There was no significant difference in time to engraftment or disease burden between vehicle or single agent ABT-199.  In contrast, less than one half of the animals treated with dasatinib engrafted by 90 days while none of the animals treated with both dasatinib and ABT-199 engrafted.   Most intriguing was the decrease in disease burden as measured by splenic size in the combination group compared to all other groups (P<0.0001, one-way ANOVA).  Analysis of BCL-2 family proteins from mononuclear cells isolated from untreated animals confirmed upregulation of BCL-2 and relatively low levels of MCL-1.  Animals treated with ABT-199 had greatly upregulated levels of MCL-1, while those treated with dasatinib or the combination did not.

Conclusions:

The combination of ABT-199 with dasatinib synergistically targets Ph⁺ALL cells both in vitro and in vivo, laying the foundation for further evaluation in vivo for adult Ph⁺ALL.  As demonstrated by others, malignancies that are particularly susceptible to BCL targeting are those which display high BCL-2 expression and a low MCL-1: BCL-2 ratio.  Combined targeted therapies may offer the potential for greater and longer responses without the morbidity associated with cytotoxic chemotherapy, particularly in older adults.