Notch-Mediated Suppression of PKCθ Reduces Reactive Oxygen Species and Promotes Leukemic Stem Cell Activity In T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Abstract 12

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cell progenitors which affects both children and adults. Current therapies achieve cure in about 80% of pediatric patients, but only 40% of adults survive beyond 5 years. Disease relapse may be attributed to ineffective targeting of leukemia stem cells (LSCs) due to their presumed quiescence, resistance to apoptosis, enhanced expression of DNA repair enzymes and drug efflux pumps, and localization to protective or inaccessible niches. More efficient targeting of LSCs in this disease thus could offer great potential for improving patient outcomes.

Recent interest in the role of reactive oxygen species (ROS) in cancer stem cell biology has been sparked by studies demonstrating that low ROS levels promote clonogenicity and radioresistance of breast cancer stem cells and that parthenolide specifically induces apoptosis of LSCs in AML and blast crisis CML by increasing levels of ROS in these cells. In T-ALL, activation of NOTCH1 signaling, either by mutation of NOTCH1 itself (50-60% of cases) or its negative regulator FBW7 (15% of cases), contributes to malignant behavior by blocking differentiation, promoting cellular proliferation/survival, and enhancing self-renewal. Similar gain-of-function Notch1 mutations are also observed to occur spontaneously in several murine T-ALL models at high frequency. To examine ROS levels in T-ALL LSCs, we first generated murine disease by retroviral transduction of bone marrow with a constitutively activated form of NOTCH1 termed “ΔE”. We utilized limiting dilution transplantation analyses of different cellular phenotypes isolated from primary ΔE leukemias to demonstrate that the CD44+ subset of CD4+ CD8+ (DP) cells were enriched for LSCs and exhibited lower ROS levels as compared to the CD44- DP subset and bulk cells. Since ROS levels were heterogeneous within the CD44+ DP subset, we combined these markers with DC-FDA staining for ROS and demonstrated as few as 100 ROS^low CD44+ DP cells are able to reproduce disease in 100% of recipients whereas 100,000 bulk cells are required to achieve the same effect.

Because protein kinase C theta (PKCθ) was recently identified as an important mediator of ROS production in T-cells during activation-induced cell death and expression of PKCθ mRNA could be detected broadly in primary human and mouse T-ALL samples, we reasoned that this signaling intermediate might be a regulator of ROS levels in malignant T-ALL LSCs. In fact, using a combination of viral expression and knock-down approaches in both human and mouse T-ALLs as well as mouse PKCθ^null ΔE leukemias, we demonstrate that PKCθ is a primary regulator ROS production in T-ALL. Moreover, low PKCθ/ROS levels correlate with resistance to chemotherapy and irradiation, and promote the competitive in vivo growth of T-ALL cells. Interestingly, we also find that Notch signaling represses PKCθ expression which in turn results in reduced ROS levels. Our results identify PKCθ as a novel target of Notch signaling in LSCs whose key malignant properties are highly dependent on low ROS levels. These findings provide mechanistic basis for rational design of LSC-targeted therapies in T-ALL.