Stathmin 1 Is Highly Expressed in Acute Promyelocytic Leukemia and Microtubule Dynamics Is a Potential Target for ATRA-Resistant APL Cells

Abstract

Introduction : Stathmin 1 is a microtubule destabilizer protein involved in the proliferation and differentiation of early hematopoietic progenitors. Aberrant Stathmin 1 expression has been reported in hematological malignancies. Stathmin 1 silencing reduces cell proliferation and clonogenicity of leukemia cell lines. A previous study identified that the PML-RARα fusion protein, which is necessary to acute promyelocytic leukemia (APL) genesis, positively regulates Stathmin 1 at transcription and protein activity levels. However, Stathmin 1 expression and its impact on prognosis and leukemia cell biology have rarely been studied in APL. Objectives : To evaluate Stathmin 1 expression and its association with laboratory and clinical parameters in a cohort of APL patients. To investigate Stathmin 1 expression and activity upon all-trans retinoic acid (ATRA) treatment and during cell cycle in the PML-RARα cell lines. Finally, to phenocopy the effects of Stathmin 1 inhibition using the pharmacological microtubule stabilizing agent paclitaxel. Methods : Bone marrow samples from 121 APL patients enrolled in the International Consortium on Acute Promyelocytic Leukemia (IC-APL) at diagnosis and 22 healthy donors were included. Patients were treated with ATRA and chemotherapy as described elsewhere. Patients were dichotomized according to Stathmin 1 expression using the median as cut-off. The PML-RARα cell lines, NB4 (ATRA-sensible) and NB4-R2 (ATRA-resistant) were used for functional assays. Cell cycle was synchronized by double thymidine block. For dose-response curves (MTT), PML-RARα cells were treated with graded concentrations of paclitaxel and IC50 values were calculated using a nonlinear regression analysis. For apoptosis assay (annexin V/PI), PML-RARα cells were treated with ATRA (1 µM) and/or IC50 paclitaxel dose for 72 hours. Gene and protein expressions were evaluated by qPCR and Western blot, and microtubule networks by confocal microscopy. For statistical analyses, Fisher's exact test or Chi-square test were used to compare categorical variables. Mann-Whitney test, t Student test or ANOVA test and Bonferroni post-test were used to compare continuous variables. Log-rank test and Cox regression were used for survival analyzes. P -value <0.05 was considerate statistically significant. Results : Stathmin 1 transcripts were significantly increased in bone marrow samples from APL patients compared with healthy donors (median 1.21 [range 0.02-4.62] versus (vs.) 0.66 [0.01-1.71]; p <0.01). Stathmin 1 levels did not impact on disease-free survival, overall survival and complete remission rates of APL patients. No relevant association between Stathmin 1 expression and clinical and laboratory parameters were observed. In NB4 cells, but not in NB4-R2, ATRA induced granulocytic differentiation, associated with a prominent reduction of Stathmin 1 gene and protein expression (p <0.05). Paradoxically, ATRA-induced granulocytic differentiation strongly reduced α-tubulin acetylation (microtubule stability marker) in NB4 cells. Using thymidine-synchronized cells, we observed that Stathmin 1 phosphorylation at serine 16 (an inhibitory site) was predominant in pool of mitosis-enriched cells, and its phosphorylation was marked reduced in pool of G1-enriched cells. NB4 and NB4-R2 cells presented high sensibility to paclitaxel treatment (IC50 of 3.6 nM and 1.8 nM, respectively). In NB4-R2 cells, but not in NB4 cells, paclitaxel increased the number of apoptotic cells after 72 hours of ATRA treatment; increased annexin V and caspase 3 cleavage (p <0.05). Paclitaxel treatment increased acetyl-α-tubulin, y-H2AX and CDKN1A levels. Conclusions : Stathmin 1 is highly expressed, but does not impact clinical outcomes in APL patients. Stathmin 1 reduces during ATRA-induced cell differentiation. Stathmin 1 activity is reduced during mitosis (as demonstrate by increased p-Stathmin 1S16) and increases post-mitosis (reduced p-Stathmin 1S16), indicating that this protein participates in microtubule dynamics and cell cycle progression in PML-RARα cells. Paclitaxel was an efficient pharmacological agent in inducing microtubule stabilization, reducing cell viability, increasing mitotic catastrophe as monotherapy or in combination with ATRA, in PML-RARα cells, including ATRA-resistant cells. Our study provides new insights on Stathmin 1 and microtubule dynamics in APL. DisclosuresBittencourt:Janssen, Takeda: Honoraria.