Class I/II Histone Deacetylase Inhibitors Are a Potential Therapeutic For Notch-1 Driven T Cell Acute Lymphoblastic Leukemia
Authors: Waibel Michaela , Hawkins Edwin D , Ramsbottom Kelly M , Martin Benjamin P , Johnstone Ricky W 
Published: October 2019

Abstract

Introduction
Although T cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease, mutations resulting in activation of the Notch-1 signalling pathway are present in over 50% of patients, thus defining Notch signaling as a central player in T-ALL disease regulation. Furthermore, despite improvement in remission rates following conventional chemotherapeutics, the prognosis for T-ALL remains poor due to disease relapse, which is often refractory to the initial therapies. Inhibitors against the γ-secretase complex, which is part of intracellular Notch-1 (ICN1) signalling, have so far shown limited efficacy and are associated with severe toxicity. Therefore, alternative approaches to treat Notch-1-driven T-ALL are required. The gene expression signature of Notch-1-mutated T-ALLs has revealed downstream activation of numerous pathways, including Myc, PI3K-AKT-mTOR and NFκB, indicating potential sensitivity to small molecule histone deacetylase inhibitors (HDACi). Therefore, the aim of this study was to use a traceable pre-clinical mouse model of Notch-1 driven T-ALL to investigate the potential of HDACi as therapy.

Methods
Retroviral transduction of mouse hematopoietic stem cells with constructs encoding ICN, followed by transplantation into irradiated recipient mice, is an established model of T-ALL resulting from Notch-1 activating mutations, and closely mimics human disease. For the present study, we transduced foetal liver stem cells from day E14.5 embryos with constructs expressing either ICN1-GFP, or GFP only. Cohorts of recipient mice developed leukaemia as described previously, characterized by splenomegaly, lymphadenopathy, elevated peripheral white blood cell counts, and the presence of double positive CD4+CD8+GFP+ blasts in peripheral blood, with a small fraction of recipients presenting with single positive CD4+GFP+ or CD8+GFP+ blasts. T-ALL blasts were isolated from this primary leukemia colony and transplanted into sub-lethally irradiated recipient mice. Using this model, cohorts of mice rapidly developed onset of leukemia with significant engraftment of T-ALL blasts in bone marrow, spleen, thymus, liver and detectable blasts in peripheral blood 15 days post transplant.

Results
GFP+ T-ALL blasts were isolated from lymph nodes and spleen and cultured in vitro in the presence of αCD3 and interleukin 2 to induce robust proliferation. Treatment of these T-ALL blasts with the pan-HDACi Panobinostat (LBH-589) induced cell death and inhibited proliferation of remaining viable blasts in a dose-dependent manner. These results illustrate that LBH-589 is a potent inhibitor of survival and proliferation of Notch driven T-ALL at low nM concentrations in vitro.
Furthermore, treatment of cohorts of mice transplanted with ICN1-T-ALL with LBH-589 significantly prevented expansion of disease in vivo as determined by white blood cell counts. Additionally, we monitored leukemic cells infiltration in secondary lymphatic tissues using non-invasive GFP imaging. Whilst high GFP readings were observed in vehicle treated groups, we observed very little signal in LBH-589 treated mice confirming prevention of disease expansion. Of most importance, the effect of LBH-589 on progression of disease was reflected in a significant increase in survival of treated cohorts compared to controls.

Conclusions
The considerably high relapse rates of T-ALL patients demands the development of novel therapies for disease that is refractory to initial therapies. Our results indicate that LBH-589 has strong potential for development of therapies for Notch driven T-ALL and may be a useful addition to current therapies, either during initial treatment or after relapse of chemoresistant T-ALL. Moreover, we have preliminary data suggesting that LBH-589 has the ability to target the predominant oncogenic “Myc signature” associated with constitutive Notch expression. We are currently performing gene and protein expression analysis to exactly determine the molecular effects of HDACi on ICN and associated downstream effector signalling.

Disclosures:
Johnstone: Novartis: Research Funding.