الفهرس 
Acknowledgements
Molecular pathways and gene expressions related to ALLOnly 14 pages are availabe for public view
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Abstract
According to Chen et al. (2018) and Wang et al. (2022), FOXO3a is a key transcription factor that mediates a variety of physiological and pathological processes, including apoptosis, proliferation, cell cycle progression, survival, and DNA damage. It acts as a relevant tumor suppressor gene (Obrador-Hevia et al., 2012). The deregulation in FOXO3a expression and/or activity can lead to various diseases, particularly cancer (Yang et al., 2021). Pang et al. (2018) illustrated that PU.1 is an ETS-family transcription factor that serves as a ‘master regulator’ of blood lineage output. It can activate multiple genes involved in B-cell receptor signaling. Deletion of PU.1 in developing B cells leads to a high incidence of B-ALL (Batista et al., 2018). Forced overexpression of PU.1 in T-cell progenitors is known to inhibit T-cell differentiation (Van Thillo et al., 2021). The present study aims at studying the expression levels of FoxO3a and PU.1 genes in BM samples of ALL patients by real-time PCR, its correlation with clinical presentation and laboratory data of these patients, and discovering if there is a relation between the expression levels of both FoxO3a and PU.1 genes to achieve a new target therapy for the disease. 1. ALL type: In this study, the number of B-ALL cases (67.9%) was more than T-ALL cases (32.1%). This is consistent with Sayınalp et al. (2020) and Chiaretti et al. (2013) as they recorded that there is an overall predominance of B-ALL in the whole cohort, while T-ALL is much less frequent. As well, Kakaje et al., (2020) studied 203 ALL patients in Syria and found that the number of B-ALL cases (79.8%) was more than T-ALL cases (20.2%). However, Mukhopadhyay et al. (2013), studied 500 ALL patients and recorded the predominance of T-ALL in Indian adolescent patients. They reported that T-ALL cases (50.4%) were more than B-ALL cases (47.6%). This discrepancy is due to the socioeconomic and environmental factors that can play a major role in the determination of pediatric ALL type as said (Rajalekshmy et al., 2011). 2-Demographic characteristics of ALL patients: Our study’s median age of ALL patients was 10 years (the age range was from 7 months to 17 years). The number of male patients with ALL was 29 (54.7%) which is more than that of female patients which reached 24(45.3%), with male: female ratio: 1.2:1. A study done in Syria on 203 patients with de novo ALL who aged 0–14 years with a median age of 5-9 years with a male predominance (60.9%) and these results agree with our results (Kakaje et al., 2020). Similarly, another study was done in Poland on 84 newly diagnosed children with ALL (median age 5 years; range 3–10; the study had 58% male and 42% female patients (Mizia-Malarz and Sobol-Milejska, 2019). In agreement with our study, Chiaretti et al. (2013) retrospectively studied the clinical-biological features of 5202 ALL patients, enrolled in Italian multicenter protocols and included 2889 males and 2313 females, with a male-to-female ratio of 1.25. Greater incidence of all leukemia in males appears to rise as males are relatively more exposed to work-related and environmental hazards, as has been submitted by some studies (Kumar et al., 2012). 3. Clinical presentation of ALL patients: The present study revealed that lymphadenopathy (79.2%), hepatosplenomegaly (64.2%) and fever (56.6%) were the most frequent clinical presentation in ALL patients. Clarke et al. (2016), Gurbuxani et al., (2021) and Kakaje et al. (2020), who studied 203 patients with de novo ALL and reported that lymphadenopathy (82.9%) and hepatosplenomegaly (73.2%) were the main presenting symptoms, agree with our study in this finding. There was a significant relation between hepatosplenomegaly in clinical data and B-ALL, as hepatosplenomegaly was more detected in B-ALL patients (80.6%) than in T-ALL patients (29.4%) (P value: 0.000). This finding is consistent with Sallman et al. (2020) as they stated that hepatosplenomegaly can be seen in about half of the B-ALL cases However, Chiaretti et al. (2013) declared and mentioned that spleen and liver enlargements were prevalently recorded in T-ALL more than in B-ALL cases. This is due to the smaller number of patients in the study (53 cases) compared to the previously mentioned study which was done retrospectively on a more significant number of ALL cases (5202 cases) with different clinical conditions of the patients. 4. Hematological data of ALL patients: A. Leucocytic count: In the present study, T-ALL patients were presented with significantly marked leukocytosis (Median: 93.8) than B-ALL patients (Median: 13.3) (P value: 0.001). Many previous studies stated that T-ALL patients are often presented with marked leukocytosis than B-ALL patients, i.e. Chiaretti et al. (2013); Sallman et al. (2020); and Kavyanjali Sharma et al. (2021). B. Hemoglobin level: There was no statistically significant difference in hemoglobin levels between both types of ALL patients. This is inconsistent with Noronha et al. (2011) and Kavyanjali Sharma et al. (2021) as they evaluated the clinical and hematological features in ALL patients (68 cases) and found that there were no significant differences in hemoglobin levels between T-ALL and B-ALL cases. Contrary to the present study, Chiaretti et al, (2013) found a significant difference in hemoglobin levels as they were <10g/dL in B-ALL patients (77.14%) compared to T-ALL patients (42.94%). The discrepancy between studies may result from the small number of patients in the present study (53 cases) compared to the previously mentioned study that was done retrospectively on a more significant number of ALL cases (5202 cases) with different clinical conditions of the patients. C. Platelets count: In the present study, a significant increase in platelet count in T-ALL patients (Median: 140) compared to B-ALL patients (Median: 22) (P value: 0.000) was found. This result is consistent with Chiaretti et al. (2013) who declared that cases with platelet count <100×109/L were observed in B-ALL cases (70.12%) compared to T-ALL cases (64.9%). However, Kavyanjali Sharma et al. (2021) evaluated the clinical and hematological features in 68 ALL Indian patients and reported that the insignificant increase in platelets count was higher in B-ALL cases than in T-ALL cases. The discrepancy between this study and previously mentioned study may be due to the different ethnic populations of the cases. D. Peripheral and bone marrow blast cells: Significant higher numbers of P.B and BM blast cells were observed in T-ALL cases (Median: 77) for P.B blast cells, (Median: 89) for BM blast cells than in B-ALL cases (Median: 50) for P.B blast cells (P value: 0.021), and (Median: 0.9) for BM blast cells (P value: 0.049) but no previous studies could be found to correlate these finding with them. (A) FoxO3a gene: 1. FoxO3a expression level: In the present study, a significant down-regulation of FoxO3a gene in ALL cases was recorded (Median (range): 0.33 (0.01 – 513.81)) compared to controls (Median (range): 1 (1 – 1)) (P value: 0.002). This result is similar to those of Mirzaie et al. (2019) and Mirzaei et al., (2016). This can be explained as, overexpression of FoxO3a in B and T cell lines inducing cell cycle arrest in the G1 phase and triggering apoptosis by induction of the cell cycle inhibitor protein, P27, and pro-apoptotic molecules FasL and Bim, respectively (Ausserlechner et al., 2013). Moreover, FoxO3a is an important target of the PI3K/AKT signaling pathway, which is hyperactivated in various types of cancers. Hyperactivation of this pathway in leukemia leads to inactivation of FoxO3a in leukemic cells through phosphorylation of FOXO3a, then FoxO3a is exported from the nucleus to the cytoplasm, where it interacts with 14‑3‑3 proteins, resulting in its proteasomal degradation and loss of function as a transcription factor and eventually tumor growth. This evidence emphasizes on FoxO3a as a tumor suppressor gene (Xie et al., 2019). In accordance to the present study, Ausserlechner et al. (2013) also reported that therapy-resistant T ALL patients show cytoplasmic localization of FoxO3a. It is also indicated that T ALL cells in these patients inactivate FoxO3a to escape apoptosis induction by TRAIL and Phorbol-12-myristate-13-acetate-induced protein 1(NOXA) gene.
Also, Dewar et al. (2011) declared that proteasomal inhibition by bortezomib can restore FoxO3 expression and cause regression of leukemia in cell culture studies and mouse models of leukemia, potentially offering an alternative therapeutic strategy in leukemias where FoxO3 is downregulated and suggesting FoxO3 as a potential therapeutic marker.
Similarly, Tang et al. (2016) reported that butein, which is a polyphenolic compound that has been identified in several plants, inhibits cellular proliferation and induce cell cycle arrest in ALL cells via activating the FOXO3a/p27kip1 pathway. Butein increases the expression of FOXO3a enhances the binding ability between FOXO3a and p27kip1 and promotes the expression of p27kip1. Additionally, butein significantly suppresses the growth of primary ALL.
Forkhead (Fox) family studies:
Forkhead box protein P1 (FOXP1) gene expression was significantly downregulated in B-ALL cases at new diagnosis (median=0.06, p<0.0001) and relapse (median=0.001, p<0.0001) phases, compared to the control group (median=0.08). FOXP1 gene expression on the 15th day of the treatment was significantly higher than its level at the new diagnosis stage (p<0.001). Moreover, the FOXP1 gene was significantly downregulated in the relapse phase compared to the new diagnosis (Tamaddon et al., 2020). As well, Zheng et al. (2020) identified that low FOXO1 transcription levels were found to be strongly associated with unfavorable ALL subtype, MRD positivity, and relapse. Furthermore, Yang et al. (2020) explored the association of FOXO3 single-nucleotide polymorphisms (SNPs) with ALL risk in Chinese children and genotyped four polymorphisms (rs17069665 A>G, rs4945816 T>C, rs4946936 C>T, and rs9400241 A>C) of FOXO3 in 425 ALL cases and 1339 health controls. They found that rs17069665 is related to the increased ALL risk but rs9400241 is related to the decreased ALL risk. Different studies supported that FoxO3a acts as a tumor suppressor gene in different types of cancers and down-regulation of FoxO3a expression can lead to malignancy: According to Yang et al. (2013), the expression of FOXO3a mRNA was lower in cancerous tissues of gastric adenocarcinoma (p = 0.03) compared with their adjacent non-tumorous tissues. Shou et al. (2012) reported that FOXO3a was low expressed in nasopharyngeal carcinoma. Zhang et al. (2021) stated that the expression of FOXO3a was lower in upper tract urothelial carcinoma patients than in normal tissues. Tian et al., (2020) demonstrated that decreased FOXO3a expression was related to increased tumor stage (P = 0.001) and grade (P = 0.035), positive lymph node metastasis (P = 0.007), and poor survival outcome in cervical carcinoma, while FOXO3a overexpression exhibited the opposite effects on cervical carcinoma. Akdeniz Odemis, (2021) found that the expression level of FOXO3a was statistically and significantly downregulated in CML patients, and AML patients compared with the expression levels in healthy controls. Zhou et al. (2019) revealed that the expression of the Foxo3 gene in de novo patients was significantly lower than in control samples in AML (P = 0.009). Contrary to the present study, Hosseini Bandari et al., (2021) evaluated the expression of oxidative stress resistance genes including Catalase, manganese superoxide dismutase (MnSOD), Foxo3a, and sirtuin-1(SIRT1) in 60 ALL patients by real-time PCR. They assumed that the expression of all studied genes were significantly higher in ALL patients than in the control group; catalase gene, FOX gene, MnSOD gene, and SIRT1 gene were expressed 4 times (p =0.04), 4.5 times (p =0.001), 2.2 times (p =0.05) and 4.8 (p =0.01) times higher than healthy individuals in the control group respectively. This discrepancy could be due to the different age groups of the patients (the age of the patients in that study ranged from 2-70 years). 2. FoxO3a expression levels between B-ALL and T-ALL cases: In the present study, there was no significant difference in FoxO3a gene expression between B-ALL and T-ALL cases. The studies of Mirzaei et al., (2016) and Mirzaie et al., (2019), which studied the expression levels of the FoxO3a gene in 70 children with different stages of ALL patients by real-time PCR, agree with the current study in this finding. They found also that the expression of FoxO3a mRNA was lower in newly diagnosed ALL patients compared to controls (P < 0.0001), maintenance (P = 0.0342), and relapse (P = 0.0006) groups. In addition, T ALL patients showed decreased expression of FoxO3a compared to the Pre B ALL one (P < 0.0001). 3. Foxo3a expression levels and age of the patients: No statistical significance between age and FoxO3a gene expression was found. This agrees with Zhou et al. (2019) who studied the expression of Foxo3 in 122 de novo AML patients by real-time quantitative PCR.
4. Foxo3a expression levels and sex of the patients: There was no statistically significant correlation between sex and FoxO3a gene expression. This result agrees with Zhou et al. (2019) but his study was done on AML patients. No other studies detected sex relation to FoxO3a expression levels in ALL patients as far as we know. 5. Foxo3a expression levels and clinical presentation of the patients: In the present study, a significant correlation between the down-regulation of FoxO3a gene expression and hepatosplenomegaly was recorded (P value: 0.042), but a correlation with this result was not examined in previous studies. 6. Foxo3a expression levels and laboratory data of the patients: There was no statistically significant correlation among white blood cells (WBC), hemoglobin, platelets, BM blast percentage and FoxO3a gene expression. This agrees with Zhou et al. (2019) who studied the expression of Foxo3 and circ-Foxo3 in 122 de novo AML patients by real-time quantitative PCR. As well, Hosseini Bandari et al., (2021) studied the expression of oxidative stress resistance genes including Catalase, MnSOD, FoxO3a, and SIRT1 in ALL patients by real-time PCR, similar to the present study, and found that there was no significant relationship between FoxO3a expression and blast percentage. In the present study, the lower expressions of the FoxO3a gene were significantly related to the higher percentage of P.B blast cells (r value: -0.284, P value: 0.048). Kornblau et al., (2010) agreed with the current study in that the higher levels of inactivated phosphorylated FOXO3a were associated with higher P.B blast cells, but differed in that the higher levels of inactivated phosphorylated FOXO3a were associated with higher WBC count and percentage of BM blast cells but in our study we did not find any significant correlations among FoxO3a expression, WBC count and percentage of BM blast cells. This discrepancy may be due to that study was done on AML patients and difference in statistical method.
(B) PU.1gene 1. PU.1 expression level: PU.1 gene expression was lower in ALL patients (Median (range): 0.09 (0.002-1.28)) than that in healthy controls (Median (range): 1(1-1)) (P value: 0.000). Similarly, Sokalski et al. (2011) ; Nakhost et al., (2019) and Batista et al., (2018) in their studies to the expression levels of PU.1 in newly diagnosed ALL patients, agree with the present study in that deletion or down-regulation of PU.1 gene can cause ALL. Batista et al., (2018) studied this finding by using RNA and whole-exome sequencing. This finding may be due to the BLNK expression as it is an important pathway through which PU.1 regulates B cell differentiation. B cells deficient in PU.1 have severely impaired BCR signaling and consequently, cannot efficiently differentiate into follicular B cells. PU.1 regulates several genes required for BCR signaling, including Btk. Interestingly, mutation of Btk strongly cooperates with mutations in BLNK to induce B-ALL in mice. These results suggest that PU.1 increases BLNK and Btk levels to enforce B cell differentiation and oppose oncogenic transformation (Xu et al., 2012). PU.1 has been implicated as an oncogene or tumor suppressor, depending on the circumstance (Xu et al., 2012). PU.1 acts as a tumor suppressor gene: Zhu et al. (2012) declared that low PU.1 expression in acute promyelocytic leukemia (APL) patients was required for disease initiation and progression. Bonadies, Pabst and Mueller, (2010 ( and (Will et al.,2015) found that PU.1 expression was reduced in AML patients. Contrary to the present study, Lin et al. (2017) who studied 80 formalin-fixed paraffin-embedded specimens with invasive breast cancer samples and assumed that the expression levels of PU.1 protein in breast cancer samples were significantly higher if it was compared with normal breast tissues by western blotting and Fluorescence in situ hybridization (FISH). This may be due to different tissue pathology and different techniques as this study is done on breast tissue by western blotting and FISH. 2. PU.1 expression levels between B-ALL and T-ALL cases: There was no significant difference in PU.1 expression between B-ALL and T-ALL, but we could not find any correlation with this result in previous studies. Seki et al. (2017) studied the role of the PU.1 gene in the occurrence of T-ALL and identified new recurrent gene fusions involving SPI1 (Recombinant Stathmin 1(STMN1)-SPI1) and (TCF7-SPI1). The positive cases for fusions involving SPI1 (encoding PU.1), account for 3.9% of the examined pediatric T-ALL cases and have uniformly poor overall survival. 3. PU.1 expression levels and age of the patients: A significant negative correlation between the PU.1 expression levels and the age of the patients was recorded (r value: -0.308, P value: 0.025), the higher expression levels were correlated to the younger age of the patients. Similarly, Zimmer et al., (2021) revealed that there is a negative correlation between PU.1 expression and the age of patients with tubular sclerosis disease (r value = −0.74, P value = 0.0064). 4. PU.1 expression levels and sex of the patients: In the present study, we did not find a significant correlation between PU.1 expression and the sex of the patients, a correlation with this result was not examined in previous studies. Several studies should be done with a larger sample size to evaluate this finding. 5. PU.1 expression levels and clinical presentation and laboratory data of the patients: According to the present study, significant correlations among the down-regulation of the PU.1 gene, hepatosplenomegaly (P value: 0.041), and the lower hemoglobin levels (P value: 0.049) were found. Kosmider and Moreau-Gachelin, (2006) and Guillouf, et al., (2006) revealed that transgenic mice were engineered to overexpress PU.1 in the hematopoietic lineages. This caused the development of erythroleukemia and forced PU.1 activity blocks the differentiation of proerythroblasts and remains under erythropoietin (Epo) control. Later on, PU.1 transgenic proerythroblasts lose their Epo requirement for proliferation and become tumorigenic. This caused the development of severe anemia and hepatosplenomegaly. There were no significant correlations among PU.1 expression, WBCs count, Platelets count and blast percentage in ALL patients. A correlation with these results was not examined in previous studies. Several studies should be done with a larger sample size to evaluate these findings. There was a significant correlation between the down-regulation of the PU.1 gene and leucocytic count in B-ALL patients (P value: 0.038) but correlations with this result were not examined in previous studies. 6. Correlation between FoxO3a and PU.1 expression levels: In the present study, there was a positive correlation between FoxO3a and PU.1 gene expression (r value: 0.326, P value: 0.017). Xie et al. (2018) agree with our study and found that both PU.1 and FOXO3 bind miR-155. PU.1 promotes FOXO3 expression by acting as a competing endogenous RNA for FOXO3 and knockdown of PU.1 decreases FOXO3 expression. The FoxO3a gene was significantly downregulated in ALL patients. Statistically significant correlations among downregulation of the FoxO3a gene, hepatosplenomegaly, and the higher percentage of peripheral blast cells were recorded. The PU.1 gene was significantly downregulated in ALL patients. Statistically significant correlations among downregulation of the PU.1 gene, hepatosplenomegaly, lower hemoglobin levels, and the older age of the patients were found. A statistically significant positive correlation between FoxO3a and PU.1 expression levels was estimated. The study concluded that the FoxO3a and PU.1genes act as tumor suppressor genes, thus rendering the FoxO3a and PU.1genes as potential targets for treating ALL as one of hematopoietic malignancy. Based on our study, we recommend the following: Studying the effect of bortezomib and butein in ALL patients, to use the Foxo3a and PU.1 genes as therapeutic targets in future therapeutic trials in ALL. Studying the expression levels of both FoxO3a and PU.1 genes at different stages of ALL (15th day after treatment, maintance, and relapse). Studying the expression levels of FoxO3a and PU.1 among pre B-ALL patients. Studying the expression levels of other members of the Fox family in ALL patients like FoxP1, FoxO1, and FoxM1. Studying the expression levels of the different genotypes of FoxO3a in ALL patients (rs17069665 A>G, rs4945816 T>C, rs4946936 C>T, and rs9400241 A>C). Studying the role of expression levels of FoxO3a and PU.1 genes as prognostic factors in ALL. Studying the expression levels of both FoxO3a and PU.1 genes by different technology as sequencing and NGS. Studying the different types of fusion genes that occur in T- ALL like (STMN1-SPI1) and (TCF7-SPI1). Studying the expression levels of oxidative stress resistance genes including catalase gene, MnSOD gene, and SIRT1 gene in ALL patients