Targeting BCL6-Mediated Resistance to BCR-ABL Targeted Tyrosine Kinase Inhibitors (TKIs) in Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL) through the Addition of Histone Deacetylase (HDAC) Inhibitors

Introduction: Patients who harbor the Philadelphia (Ph+) chromosome t(9;22) translocation account for approximately 20-30% of adult ALL and 2-5% of pediatric ALL. Prior to approval and use of imatinib, a small molecule TKI which targets the Ph+ chromosome BCR-ABL1, these patients had poor survival & EFS - with long term survival rates in the 20% range. With the addition of imatinib and later generation TKIs to chemotherapy backbones and bone marrow transplant, EFS & survival rates have substantially improved - surpassing 50% in studies in adults and even higher in children. However, resistance to imatinib and other TKIs has become a significant problem in Ph+ ALL, especially in adults. ABL1 kinase domain mutations are the dominant form of TKI resistance, however other resistance mechanisms include upregulation of parallel pathways such as SRC family kinases, MAPK and BCL6 pathways. BCL6 is an oncogene that suppresses transcription of tumor suppressor genes such as p53 and CDNK1A. Interestingly, BCL6 has been shown to be upregulated and activated through deacetylation following imatinib treatment in Ph+ ALL, likely leading to its role in resistance. Histone deacetylase inhibitors (HDACi) have been shown to act synergistically with TKIs in imatinib sensitive and resistant Ph+ leukemia though multiple mechanisms including attenuation of BCR-ABL1 levels and other downstream proliferation promoting pathways. We have shown that HDACi treatment acetylates (and thus inactivates) BCL6 in Ph+ ALL, and that the combination of HDACis and TKIs leads to synergistic effects in vitro and in vivo (using xenograft models).

Methods: In vitro WST-1 cell viability assays were carried out on TOM1 cells (non-ABL1 mutant, imatinib sensitive Ph+ ALL) and NALM1 cells (non-ABL1 mutant, imatinib resistant CML lymphoid blast crisis) with imatinib and entinostat (a HDACi). Synergy was assessed using Calcusyn software. Western blots were performed assessing BCL6 expression and acetylation, and expression of downstream effectors of apoptosis. Two separate in vivo xenograft mouse experiments were performed transplanting TOM1 and NALM1 cells into Nod SCID Gamma (NSG) mice. Cohorts of TOM1 mice were treated with imatinib 50mg/kg BID, entinostat 15mg/kg QD, imatinib plus entinostat combination, or vehicle control. In the NALM1 mice we added a higher dose imatinib cohort (100 mg/kg BID) due to known imatinib resistance.

Results: In vitro, there was substantially more synergy of the imatinib/entinostat combination in imatinib-resistant NALM1 cells vs. the imatinib-sensitive TOM1 cells. Average Combination Index (CI) values in TOM1 cells across multiple entinostat and imatinib doses was 1.2 (CI: =1 suggest additive effect, <1 = synergy, >1 = antagonism), while the CI in NALM1 cells at the same dose combinations was 0.53. We noted BCL6 upregulation and decreased BCL6 acetylation - signs correlating with resistance - in Western blots of NALM1 and TOM1 cells treated with imatinib, while exposure to entinostat caused increased acetylation of BCL6 and increased expression of downstream tumor suppressors.

In the imatinib-sensitive TOM1 xenograft trial, the combination displayed a significant reduction in bone marrow leukemic blast involvement versus control following 6 weeks of dosing as measured by flow cytometry (36.9% mean decrease, p=0.001). There was a trend toward decreased bone marrow involvement between the combination treatment and other active treatment arms. There was no difference in peripheral blood blast percentage between arms. In the imatinib-resistant NALM1 xenograft trial, the combination showed a significant decrease in peripheral blood blast percentage in the combination arms versus all other arms after only two weeks of therapy (p=0.0008).

Conclusions: Upregulation of activated BCL6 is a known mechanism of resistance in Ph+ ALL that may be abrogated by acetylation of BCL6 with HDACi, as our in-vitro data suggests. Further, we have shown in xenograft models of Ph+ acute lymphoblastic leukemia that combination therapy with HDACi + imatinib, even in imatinib-resistant leukemia, has significant activity. Interestingly, the combination appears more active in resistant disease than in imatinib-sensitive disease. This combination could prove a viable strategy to attenuate imatinib- (and perhaps other TKI-) resistance in Ph+ ALL relapse, particularly in cases not driven by ABL1 kinase domain mutations.