In Vitro Colony Growth, RAS Genes Sequencing, and Expression of Bcl-2 and Bax Proteins in Chronic Myelomonocytic Leukemia.
Authors: Onida Francesco , Ricci Clara , Servida Federica , Fermo Elisa , Molteni Mauro , Cesana Clara , Cortelezzi Agostino , Soligo Davide , Lambertenghi-Deliliers Giorgio 
Published: April 2020

Abstract
Chronic myelomonocytic leukemia (CMML) is a heterogeneous hematological malignancy, which shows features of both myelodysplastic and myeloproliferative disorders. It has poor prognosis due to the lack of effective treatment and very little is known about its pathogenesis. A point mutation in the RAS genes is detected in 20 to 35% of patients with CMML, with higher percentage among patients with proliferative variant of the disease (MP-CMML, WBC &gt; 13 x 109/L). The expression of Bcl-2 family proteins, which are key regulators of the mitochondrial-mediated pathway of apoptosis, have been reported as being altered in MDS and other hematological disorders. However, the role of these proteins has not been systematically investigated in CMML.
In this study, we evaluated by Western Blotting the expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins in peripheral blood mononuclear cells (PBMNC) from 21 CMML patients and 8 age-matched healthy controls. We also investigated the presence of point mutations within codons 12, 13, and 61 of N- and K-RAS by sequencing analysis, and assessed in vitro colony growth of PBMNC with and without growth factors (GFs) [SCF, GM-CSF, IL-3, Epo]. Our series included 10 males and 11 females, with a median age of 74 years. Median WBC value was 9.2 x 109/L (range 3.2 to 17.2), while differential showed median monocyte values of 27.5% and 2.0 x 109/L. Six out of the 21 patients had MP-CMML. All patients but one had normal karyotype. A point mutation within N-RAS (G12D) was detected in two patients, both with MP-CMML. Spontaneous growth was observed both in normal controls and in CMML (median 19 and 14 CFU-GM/105 MNCs, respectively), with a higher number of CFU-GM in the MP-CMML than in the dysplastic subgroup (MD-CMML) (46 vs 12). After addition of GFs, we observed an increase of colony number in all groups. Interestingly, in vitro colony growth was remarkably higher in the two patients with mutant N-RAS than in patients with wild type RAS, especially after the addition of GFs (200 vs 57, p &lt;0.05). The expression of Bax was higher in CMML than in the control group (3.81 vs 1.39, p = 0.07), whereas Bcl-2 was lower (0.88 vs 1.41, p = .16), even though these differences were not statistically significant. No differences were observed between subgroups of patients with MD- and MP-CMML. When the Bcl-2/Bax ratio was calculated, we observed lower values in CMML than in normal controls (0.27 vs 1.08, p &lt;0.01). In the two patients with mutated RAS, Bcl-2 expression was significantly higher than in patients with wt-RAS (2.07 vs 0.75, p &lt;0.05). Noteworthy, Bcl-2 expression in patients with mutant RAS was also higher than in the control group. In conclusion, our data suggest a deregulation of mechanisms controlling apoptosis in CMML, with no significant differences between MD and MP variants. The finding of higher Bcl-2 expression in patients with mutant RAS suggests a possible cooperation of this anti-apoptotic protein with RAS-activated intracellular pathways, warranting further confirmation in larger CMML series. In agreement with previous reports, we observed higher in vitro spontaneous growth in MP-CMML than in MD-CMML. Hypersensitivity to GFs (possibly GM-CSF) in cells from patients with mutant RAS is suggested and merits further investigation.