Inactivation of INK4a/ARF Accelerates the Development of PTEN Deficient T-ALL
Authors: Yang Yaling , Gao Xiuhua , Han Xin , Huang Xiaoyan , Liu Qing , Zhang Yu H. , Yin C. Cameron , Chen Ling , You M. James 
Published: October 2019

Abstract
Abstract 4197

Introduction:
Acute T-lymphoblastic leukemia/lymphoma (T-ALL) is a group of heterogeneous disease with deregulated expression of variable types of genes. PTEN and INK4a/ARF are among the most frequently inactivated tumor suppressors in human T-ALL. The PTEN gene encodes a phosphatase that negatively regulates the phosphatidylinositol 3-kinase (PI3K) pathway activity. PI3K activates a variety of key signaling proteins that are critical in cell cycle control and cell survival. The Ink4a/Arf gene encodes two distinct tumor suppressors, p16 INK4a and p14 ARF (p19ARF in mice), that function as regulators of the pRB and p53 pathways, respectively. Since dual inactivation of PTEN and INK4a/ARF tumor suppressor genes occurs relatively frequent in human T-ALL, we investigated the specific role and inter-relationship of PTEN and INK4a/ARF tumor suppressor genes in the pathogenesis of T-ALL in genetically engineered mouse models (GEMMs).

Methods:
We have intercrossed the Ptenflox/flox mice with Ink4a/Arfflox/flox mice, generating compound conditional KO mice carrying both Ptenflox/flox and Ink4a/Arfflox/flox alleles. The compound Ptenflox/flox and Ink4a/Arfflox/flox mice were further intercrossed with Lck-Cre mice, an extensively characterized transgenic mouse strain that expresses Cre recombinase under the T-cell specific LCK promoter. The Lck-Cre transgenic mice express Cre in T-lymphoid cells, including T-lymphoid cells at early developmental stage. Serial analyses from birth to 8 months of age (9 time points) and/or end-point study were performed for a phenotypic workup. Kaplan-Meier curves for overall and tumor-free survival were generated at the end of the study. Grossly normal or tumor-bearing tissue was harvested for histopathlogical and molecular analyses.

Results:
Molecular analyses showed expected recombinant alleles and absence of Pten and INK4a/ARF proteins in mouse embryonic fibroblasts (MEFs) and T-cells after Cre-mediated excision with concurrent increased expression of phosphorylated Akt indicating the proper deletion of the Pten tumor suppressor gene. Mice with T-cell specific deletion of either Pten, Ink4a/Arf or Pten and Ink4a/Arf were closely monitored closely for perturbations in the hematolymphoid system. The mean disease-free survival (lack of palpable adenopathy) of tInk4a/Arf-/- and tPten-/-, littermates averaged 53, 30 weeks, respectively, while compound and tInk4a/Arf-/- and tPten-/- mice succumbed between 6 and 24 weeks (mean 12 weeks; p<0.001) to a rapidly progressive disease characterized by generalized wasting, mediastinal mass and enlarged lymph nodes. Histologically, the neoplastic cells are immature lymphoid cells, involving multiple organs and circulating blood. Immunophenotypic studies by flow cytometry demonstrated immature T-cell lineage cells (T-ALL).
Conclusions: Using our genetically engineered mouse models, we have provided genetic evidence that INK4a/ARF and PTEN cooperate in constraining the development of T-ALL. These mouse models provide a platform for discovering new molecular targets for T-ALL therapy preclinically. Ongoing efforts focus on further charactering the difference in pathology, the PI3K/AKT pathways, DNA damage, cell cycle control, and drug sensitivities among the various T-ALL models.

Disclosures:
No relevant conflicts of interest to declare.