First-in-Class HAT Activator (YF2) Combined with JAK/STAT Inhibitor (ruxolitinib) Unveils Potential Novel Treatment Approach for HDAC Inhibitor-Resistant CTCL

T-cell lymphomas are both aggressive and challenging to treat. This is in-part due to multiple epigenetic derangements found in T-cell lymphomas. One treatment strategy includes HDAC inhibitors; however, the duration of response is short for most patients which is consistent with many treatments for T-cell lymphoma. As a result, alternative treatments are required. YF2, a first-in-class histone acetyltransferase (HAT) activator, was invented at Columbia University. YF2 increases acetylation of histones by augmenting the activity of HAT enzymes.

The JAK/STAT pathway contributes to CTCL lymphomagenesis through a number of cellular processes including cell cycle, proliferation, apoptosis, etc. JAKs activate STAT3, which activates the transcription of the anti-apoptotic Bcl-2-like protein, Bcl-XL. Additionally, HATs acetylate the pro-apoptotic p53 protein, increasing its stability and activity. Given the dependence on both the JAK/STAT pathway and epigenetic derangements in CTCL, we hypothesize that if the JAK inhibitor, ruxolitinib, induces apoptosis via modulation of Bcl-XL, and YF2 induces apoptosis via acetylation and activation of p53, then dual targeting of these complementary pathways could lead to synergistic apoptosis in CTCL.

To study this hypothesis, we used two CTCL cell lines: HH [JAK wildtype (JAK ^WT)], and H9 [JAK1/3 gain-of-function mutated (JAK1/3 ^GOF)]. In addition, we generated a belinostat-resistant H9 cell line (H9-belino-R) by incrementally exposing H9 to increasing concentrations of belinostat. H9-belino-R resistance was observed at a 20-fold (IC₅₀ = 896.7nM, SEM ± 185.3) increase over that of the parental H9 (IC₅₀ = 43.8nM, SEM ± 4.61) (figure 1). H9-belino-R retained significant resistance to other HDAC inhibitors such as romidepsin [(H9: IC₅₀ = 0.97nM (SEM ± 0.030), H9-belino-R: IC₅₀ = 1.38nM (SEM ± 0.028)] and panobinostat [H9: IC₅₀ = 3.11nM (SEM ± 0.19), H9-belino-R: IC₅₀ = 9.00nM (SEM ± 1.55)] as measured by the CellTiter-Glo Viability Assay. Interestingly, the IC₅₀ of the HAT activator, YF2, was retained between the resistant (14.84µM, SEM ± 0.46) and parental (12.45µM, SEM ± 0.27) cell lines (figure 1).

HH (JAK ^WT) showed significant cytotoxicity (63.5% live, 30.7% apoptotic, and 5.4% dead) after 4-day exposure to 10µM YF2 compared to limited cytotoxicity (93.3% live, 4.6% apoptotic, and 2.0% dead) in H9 (JAK ^GOF) as measured by flow cytometry with Annexin V. At baseline, active STAT3 (pSTAT3) was not detected in HH possibly contributing to its susceptibility to YF2 as the absence of pSTAT3 reduces the transcription Bcl-XL. As illustrated by figure 2, western blot analysis confirmed ruxolitinib-induced inhibition of active STAT3 (pSTAT3) with associated reduction in Bcl-XL expression in H9 and H9-belino-R. This may explain the observed synergistic cytotoxicity with YF2 and ruxolitinib in HH, H9 and H9-belino-R (Excess over Bliss scores: 4.85, 14.23, and 5.38 respectively) as determined by flow cytometry.

In conclusion, the novel first-in-class HAT activator, YF2, retains activity in HDAC inhibitor-resistant CTCL. YF2’s activity may be blunted by JAK1/3 ^GOF mutation; however, when combined with the JAK-inhibitor ruxolitinib, the response mirrored that of the HH (JAK ^WT) cell line. The combination of HAT activator and JAK inhibitor led to enhanced reduction of the anti-apoptotic protein Bcl-XL, potentially explaining synergistic apoptosis in CTCL. These preliminary findings provide us with evidence that suggests that the combination of YF2 and ruxolitinib can serve as a novel treatment combination for CTCL. Further study is planned to explore this treatment in murine models of disease.