Inhibition of the JAK/STAT and Bcl-2 Pathways Enhances Anti-Tumor Effects in an in Vitro model of T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Abstract

T cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematologic malignancy comprising 15% of pediatric and 25% of adult cases of ALL. With current treatment options, T-ALL survival rates have reached 50-60% in adults and 85% in children. Despite great strides in the treatment of this subset of ALL, T-ALL still shows resistance to first-line therapies in over 50% of adults and 25% of children, and relapse is often chemorefractory. Mutations in the Janus Activating Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathways and overexpression of the B cell lymphoma-2 (Bcl-2) protein are highly linked to the progression of T-ALL, and to the resistance of a number of available treatments for lymphoid malignancies. JAK/STAT is one of the main cytokine signaling pathways involved in hematopoietic cell growth and development. Indeed, the JAK/STAT pathway is often constitutively activated by T-ALL. The Bcl-2 pathway also plays an important role in cell survival. Overexpression of Bcl-2, an anti-apoptotic member of the Bcl-2 family, promotes cell survival by binding and neutralizing pro-apoptotic members. Inhibition of key proteins in both of these pathways has been greatly explored individually, but little is known about their combined effects on T-ALL. It is hypothesized that T-ALL manipulates both of these pathways as a means of escaping individual inhibition of either JAK/STAT or Bcl-2. Our hypothesis is that inhibiting both the JAK/STAT and Bcl-2 pathways with two small molecule inhibitors; Ruxolitinib (JAK 1/2 inhibitor) and Venetoclax (BH3 mimetic targeting Bcl-2), will inhibit T-ALL growth and survival. Proliferation of T-ALL was assessed by MTT assay and viability was measured by trypan blue and flow cytometry at 24, 48 and 72-hour time points post-treatment. Single-drug dose responses were conducted for both inhibitors. Six doses of both Ruxolitinib and Venetoclax were tested from a range of 0.156uM-5uM for Ruxolitinib and 1.56nM-50nM of Venetoclax. A response was seen for the three highest doses of both inhibitors (1.25uM, 2.5uM, and 5uM for Ruxolitinib and 12.5nM, 25nM, 50nM for Venetoclax). However, a synergistic effect was only achieved when combining 1.25uM Ruxolitinib with 25nM Venetoclax or 2.5uM Ruxolitinib with 12.5nM or 25nM Venetoclax. The combination dose of 1.25uM Ruxolitinib and 25nM Venetoclax demonstrated the greatest combined synergistic effect (CI<1) for all three assays at both 48 hours and 72 hours post-treatment. This optimal in vitro dose of 1.25uM Ruxolitinib and 25nM Venetoclax significantly lowered proliferation and viability of jurkat cells compared to no treatment (P<0.0001), vehicle control (P<0.0001) and the single-drug dose control groups (P<0.0001). Targeting both the JAK/STAT and Bcl-2 pathway with orally available FDA approved small molecule inhibitors could provide a novel alternative treatment for patients who relapse, fail or are resistant to first-line chemotherapeutic regiments. Disclosures No relevant conflicts of interest to declare.

STAT5 inhibition induces TRAIL/DR4 dependent apoptosis in peripheral T-cell lymphoma

“…Targeting the JAK/STAT and apoptotic pathways in combination presents a promising therapeutic approach in PTCL. Combination therapies with BCL-2/BCL-xL inhibitors, targeting the intrinsic pathway, and JAK inhibitor ruxolitinib have demonstrated synergy in pre-clinical models of adult T-cell leukemia lymphoma and T-ALL [ 25 , 56 , 57 ]. Our research suggests that targeting the extrinsic pathway may offer a mechanistic advantage and implicates a role for combination of STAT5 inhibition with death receptor signaling agonists as a future therapeutic approach in PTCL, although effective TRAIL agonists are still in development [ 58 ].…”

Anatomy of a crime: how IL7R and NRAS join forces to drive T-cell acute lymphoblastic leukemia

“…Roughly 10% of T-ALL patients display IL7R gain-of-function mutations, and a much larger fraction (some 50-80% of the cases) express IL7R and may benefit from IL7 produced in the leukemia milieu. [3][4][5][6][7] IL7R-mediated signaling (because of IL7, high IL7R levels or mutational activation of the receptor or downstream effectors) can promote T-ALL establishment and maintenance, and resistance to glucocorticoids. 5 RAS activating mutations in general occur in around 2% of the cases, and NRAS alterations are infrequent.…”