Acute myeloid leukaemia (AML) is a malignant disease of haematopoietic stem cells characterized by uncontrolled clonal expansion of myeloid progenitors with subsequent bone marrow failure and impaired normal haematopoiesis. Despite advances in diagnosis and treatment, AML still has a high mortality rate [10].
Autophagy is a cellular process by which waste macromolecules and excess or damaged organelles are engulfed within autophagosomes. These autophagosomes then fuse with cytoplasmic lysosomes for degradation and recycling. Thus, efficient and strict regulation of autophagy is crucial for safeguarding cell homeostasis. Nonetheless, aberrant autophagy may underlie different human pathological conditions, as demonstrated by several studies [11,12,13,14].
The BECN1 gene encodes the Beclin-1 protein, which is essential for autophagy initiation and regulation [15, 16]. Additionally, Beclin-1 is implicated in autophagic programmed cell death. In different types of human cancers, a reduction in BECN1 has been associated with accelerated tumour growth, metastasis, and poor prognosis [2].
This work aimed to assess the expression status of the BECN1 gene in newly diagnosed adult AML patients in comparison to a healthy control group and to determine its relationship to various haematological parameters and clinical outcomes, especially response to induction chemotherapy. In our study, we measured expression of BECN1 in 50 de novo AML patients and 20 healthy controls.
The AML patients showed a highly significantly reduction in BECN1 expression compared to the control group. Upon detailed analysis, we found that 72.0% of the patients had reduced BECN1 gene expression compared to the control group and that 28.0% of the patients had normal BECN1 gene expression. This result is in accordance with Zare-Abdollahi et al. [17], who analysed the expression status of the BECN1 gene in a series of 128 de novo AML patients using real-time quantitative polymerase chain reaction, in which BECN1 reduced expression was detected in 57 of the 128 cases (45%). In another study performed by Mohamadimaram et al. [18], expression of other autophagy genes (namely, ATG7 and light chain 3 (LC3)) was investigated, and the authors found a significant decrease in expression levels of both genes in most AML patients (81.81% and 75.55%, respectively). However, BECN1 was not examined.
In contrast, Keyvan et al. [19] assessed changes in BECN1 gene expression in blood samples from 30 AML patients compared with samples from 15 healthy persons and found no significant differences in BECN1 gene expression between patients with AML and normal controls (P > 0.05). This may be due to the smaller sample size in their study or to genetic differences. In addition, Tandel et al. [20] reported the reverse of our finding: BECN1 gene expression levels were significantly higher in AML patients than in controls at a rate of 5/3-fold, with a significant P value (P < 0.0001). Thus, more effort is needed to investigate the expression status of the BECN1 gene.
In AML patients, reduced BECN1 gene expression was associated with older age and higher TLC. Our values were very close to the findings of Zare-Abdollahi et al. [17], who reported a mean age of 52.9 years and a mean TLC of 52.6 × 103/μl in AML patients with reduced BECN1 gene expression. However, we did not observe a significant relationship between the level of BECN1 gene expression and sex, haemoglobin level, or platelet count. This was in accordance with Lian et al. [21] and Zare-Abdollahi et al. [17].
Furthermore, we noted that a higher percentage of AML patients in the FAB groups (M1 and M2) also showed reduced BECN1 gene expression, though this association did not reach significance. In parallel, Folkerts and colleagues [22] found no significant difference in expression levels of autophagy genes (including BECN1) among AML patients in different FAB subtype groups.
Interestingly, in our study, the FLT3-ITD mutation was detected in 38.9% of patients, and all of them had significantly reduced BECN1 gene expression. Similarly, Tao et al. [23] reported a remarkable association between reduced BECN1 gene expression and FLT3-ITD mutation. The same finding was also reported by Zare-Abdollahi et al. [17], whereby reduced expression of BECN1 coincided with the FLT3-ITD mutation in 27 of the 128 cases (21%) investigated.
Our results, along with other reports, suggest an inverse correlation between BECN1 gene expression and the presence of the FLT3-ITD mutation. A recent study showed that FLT3-ITD protects leukaemic cells treated with PI3K/Akt/mTORC1 pathway inhibitors, a common pathway dysregulated in AML [24, 25], from apoptosis by STAT5 activation and subsequent MCL1 expression induction [26]. As mTORC1 acts as an autophagy repressor, it is speculated that major regulators/initiators of autophagy, such as BECN1, can also be regarded as a potential target of FLT3-ITD or STAT5 or other yet-to-be-identified mediators [17].
In another study, FLT3-ITD mutations were found to induce an increase in basal autophagy in leukaemic cells through a previously uncharacterized signalling cascade involving the transcription factor ATF4. Moreover, inhibiting autophagy or ATF4 significantly impaired FLT3-ITD leukaemic cell proliferation as well as tumour burden in murine xenograft models. Importantly, autophagy inhibition also overcame FLT3 inhibitor resistance due to the FLT3-TKD mutation both in vitro and in vivo. These results suggest that targeting ATF4 or autophagy in AML patients carrying FLT3 mutations may represent a promising alternative therapeutic strategy [27].
A higher percentage of c-KIT mutations was found in AML patients with reduced BECN1 gene expression in our study. However, this was statistically insignificant. Similarly, the group with reduced BECN1 gene expression had a significantly higher number of patients expressing CD117, as based on flow cytometry. Considering its value in predicting a poor prognosis, the association of c-Kit mutations adds to the value of reduced BECN1 expression.
Lian et al. [21] studied the mRNA levels of BECLIN1 and ATG5 in 101 newly diagnosed leukaemia patients and reported that AML samples with CEBPα or c-KIT mutations showed lower BECLIN1 expression levels than those without mutations. Additionally, patients with c-KIT mutation exhibited lower ATG5 expression.
In our study, BECN1 gene expression was reduced in patients with intermediate and adverse cytogenetic risks compared to both favourable risk and control groups. These findings were similar to those reported by Zare-Abdollahi et al. [15]. Similarly, Marconi et al. [28] reported reduced expression levels of key autophagy regulatory genes, including BECN1, in an adverse cytogenetic risk group.
Patients showing reduced BECN1 gene expression in our study also displayed significantly higher CD34 expression by flow cytometry. These findings are similar to those reported by Watson et al. [26], who examined the status of the autophagy pathway in human AML using bone marrow (BM) samples. They sorted the populations identified by CD34 (CD34-positive blasts) and examined expression of key autophagy genes, including BECN1. They reported decreased expression of autophagy genes, including BECN1, in the CD34-positive AML blast cell population compared with the blast marker-negative population.
Interestingly, low BECN1 gene expression was highly associated with resistance to therapy in our patient cohort. Similar results were reported by Marconi et al. [28], who found that reduced expression levels of key autophagy regulatory genes (including the BECN1 gene) are linked with resistance to therapy.