NOTCH2 Contributes to Venetoclax Resistance in Chronic Lymphocytic Leukemia
Authors: Fiorcari Stefania , Maffei Rossana , Atene Claudio Giacinto , Martinelli Silvia , Potenza Leonardo , Luppi Mario , Marasca Roberto 
Published: November 2019

BACKGROUND:
Chronic lymphocytic leukemia (CLL) has recently experienced an unprecedented revolution thanks to the discovery of crucial pathogenetic mechanisms. Despite considerable therapeutic advancements, the eradication of the disease is not complete and resistance and transformation may occur. Venetoclax is a small-molecule BH3 mimetic that competes for binding in the hydrophobic groove of Bcl-2, a key protein involved in CLL cells survival. Venetoclax was shown to have an excellent antitumor activity in patients with relapsed CLL including those with high risk features. However, venetoclax monotherapy shows an overall response rate of 79% and complete response rates of 16% and leave some open questions mainly related to intrinsic features of CLL patients that may guide a pattern of resistance. A recent work shows that durable response to venetoclax based therapy is more likely in patients having minimal adenopathy, mutated IGHV gene, wild type TP53 and Notch1 genes.
AIM OF THE WORK:
We hypothesize that specific intrinsic features of CLL cells may contribute to drive possible mechanisms of resistance to venetoclax (ABT-199) treatment.
METHODS:
Notch2 expression was monitored by western blot in purified CLL cells. Modulation of Notch2 expression in vitro was performed by using siRNA strategy. ABT-199 was used at doses of 0.1 nM or 1 nM.
RESULTS:
Notch signaling is relevant in CLL pathogenesis. We detected a peculiar high expression of Notch2 in a subgroup of CLL patients carrying trisomy 12. The high expression of Notch2 correlated with high levels of Mcl-1, CD23 and Hes1. Interfering with Notch2 expression in trisomy 12 CLL patients, by siRNA silencing, was able to affect leukemic cells viability, reducing CD23 and Mcl-1 expression. Since Mcl-1 is involved in ABT-199 resistance, we wondered if ABT-199 may have a different effect in CLL cells isolated from patients carrying trisomy 12 comparing to no trisomy 12 cases. After 24h of culture in complete medium, trisomy 12 CLL cells showed a gain in survival rate of 10% during treatment with both 0.1 nM or 1 nM in comparison to no trisomy 12 cases. This advantage in viability reflected the maintenance of Notch2 and Mcl-1 expression in trisomy 12 both at 0.1 nM and 1 nM of ABT-199 compared to control. Conversely, a reduction of Notch2 and consequently Mcl-1 levels were observed in no trisomy 12 cases at both doses of ABT-199. We did not detect any variation in Bcl-2 levels. We wondered if Notch2 expression may reduce the response to ABT-199 in trisomy 12 CLL. To demonstrate this hypothesis, we interfered with Notch2 by silencing strategy. Treatment with Notch2 siRNA decreased the expression of Notch2 and Mcl-1 in combination with ABT-199 as shown in Figure 1A. We also found that Notch2 down-regulation cooperated with ABT-199 decreasing CLL cells viability (Figure 1B).
CONCLUSIONS:
Collectively, our results show a novel mechanism that may compromise the clinical response to venetoclax in CLL patients. Although the excellent mechanism of action of venetoclax, Bcl-XL and Mcl-1, two major anti-apoptotic proteins of Bcl-2 family not inhibited by venetoclax, are key determinants of both acquired and intrinsic resistance to treatment. The possibility to identify patients with more pronounced expression of Mcl-1 may help to optimize the treatment. The expression of Notch2 identify a subset of CLL patients, mainly harboring trisomy 12 aberration, that through the maintenance of high levels of Mcl-1, may be involved in a reduced response to ABT-199.

Disclosures
Luppi: Gilead Sci., MSD, Pfizer, Novartis, Abbvie, Sanofi, Daiichi Sankyo, Jazz Pharmaceuticals: Honoraria. Marasca:Janssen and Gilead Sci, Abbvie, Roche and Shire: Honoraria, Research Funding.