Primary Acute Lymphoblastic Leukemia Cells Strictly Require Interphase Microtubules for Survival during G1 Phase Advance

Abstract

Microtubule targeting agents (MTAs), such as vincristine (VCR), are important anticancer agents and are thought to act primarily in mitosis. However, there is increasing evidence that MTAs can also cause death by interfering with interphase microtubules. We have recently discovered that when primary acute lymphoblastic leukemia (ALL) cells are treated with vincristine, cells in G1 phase die directly without advancing to other phases of the cell cycle. This system represents a powerful model for understanding interphase mechanisms of MTA action, and provides an opportunity to test whether MTAs can be combined with agents that target interphase processes in order to create novel treatments for ALL. One such candidate is palbociclib (PCB), a highly selective inhibitor of cyclin dependent kinases 4/6 (CDK4/6), whose activity is necessary for the transition from G1 to S phase, providing a rationale for the combination of palbociclib and vincristine to target ALL cells in G1 phase. In this study, we tested the effect of PCB alone or in combination with vincristine in primary ALL cells. PCB caused arrest of primary ALL cells in G1 phase, with 97–99% of cells exhibiting 2N DNA content, while having no significant effect on cell viability. In contrast, HeLa cells were unaffected by PCB, consistent with their lack of dependence on the CDK4/6‐retinoblastoma pathway. When ALL cells were pretreated with PCB, they became refractory to VCR treatment. Examination of the microtubule network by immunofluorescence microscopy in ALL cells treated with PCB alone or PCB followed by VCR treatment showed that PCB did not disrupt the microtubule network, nor prevented VCR from doing so. Furthermore, ALL cells treated with ABT‐263, a Bcl‐2/Bcl‐xl inhibitor, after PCB pretreatment still underwent cell death, indicating that PCB did not block downstream apoptotic pathways. Experiments assessing entry and passage through S phase using 5‐ethynyl‐2′‐deoxyuridine incorporation showed that G1 arrest in primary ALL cells by PCB was reversible, and that cells released from arrest regained susceptibility to VCR. Taken together, these results indicate that ALL cells must be cycling in order for interphase death induced by vincristine to occur, and in turn indicate that interphase microtubules play a fundamental role in survival of actively cycling ALL cells. In addition, these findings strongly caution against combining vincristine and CDK4/6 inhibition as a potential therapy for ALL.Support or Funding InformationChancellor's Circle AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.