The majority of AML patients still succumb to this disease because of refractoriness to therapy or relapse, and new therapeutic approaches for AML are an urgent clinical need. Our study provided direct evidences that FHL1high predicted adverse outcomes for AML. Firstly, FHL1 was widely expressed at high levels in drug resistant cell lines (Fig. 1b). Meanwhile, quantitative RT-PCR was confirmed consistently that FHL1 as an important gene was differed dramatically and expressed at high level in drug resistant group (Fig. 1c). All these supported that FHL1 was a vital drug resistance gene and significantly overexpressed.
Secondly, for better studying the correlation between FHL1 expression and the prognosis of IP-AML, including some pretreatment molecular characteristics, the prognostic value of FHL1 was further confirmed in the independent group. In these 145 IP-AML patients, we found that FHL1high was linked to the presence of other adverse prognosticators. With regard to molecular factors, FHL1high patients had a lower prevalence of FLT3-ITD, NPM1 and Biallelic CEBPA mutation and higher prevalence mutation of RUNX1, higher expression levels of BAALC and MN1 than FHL1low patients (Table 1). These findings indicated that FHL1 might play an active role in leukemogenesis just like other tumor markers in IP-AML patients.
Further, patients with FHL1high were significantly more classified in the FAB M0 subgroups than with FHL1low, suggesting that the leukemic FHL1 patients derived from relatively more minimally differentiated IP-AML patients (Table 1), which might indicate adverse malignancy. In addition, based on TCGA cohorts, FHL1high was proved to have significant associations with adverse outcomes. FHL1high was associated with shorter OS, EFS and RFS, noteworthy difference in OS and EFS (Fig. 2a- c, Fig. 3). This study demonstrated that FHL1 might play an oncogenic role in leading to adverse prognosis in the development of leukemia. Meanwhile, negative factors (Age, WBC and FLT3, TP53, CEBPA mutations) were associated with poor OS and EFS. After adjusting for known prognosticators by multivariable analyses, the association of FHL1high with adverse OS and EFS still existed and FHL1high expressers harbored poor OS and EFS. In addition, the nomogram based on the model exhibits an impressive performance and clinical applicability. Moreover, prognostic significance in FHL1 was further consistently validated in another larger 284 IP-AML cohort ((Fig. 3). All these results proved FHL1 as a potential prognosticator and therapeutic target of IP-AML, which could promote further fine stratification of NCCN cytogenetically IP-group.
Disease recurrence occurs in most IP-AML patients within 3 years after diagnosis. A short duration of remission (i.e., < 6 months), adverse genetic factors, older age, and poor general health status are the major determinants of outcome after relapse [22]. Ignoring the cause of induction death by intense chemotherapy by AML patients whose survival was > 30 days, we also found that high expression of FHL1 was still independently associated with short OS and EFS. Taken together, clinical characteristics of FHL1high expressers implied FHL1 expression level might be associated with a poor outcome and results supported highlight FHL1 functions acting as a potent oncogenic role in leukemia development. Above all, these also showed that FHL1 acted as an independent adverse prognostic biomarker, which might substitute other adverse factors and might be employed to improve risk stratification of NCCN IP-patients.
Some studies also found that FHL1 in mRNA was up-regulated during embryonic skeletal muscle development [23], human hypertrophic cardiomyopathy and cardiovascular system [24]. Meanwhile, FHL1 was noticeably down-regulated in a variety of cancers including lung [25], breast [26, 27], colon [28] and gastric cancers [29]. This paper supported that FHL1 was up-regulated and acted as a potent oncogenic role in leukemia development. Here, we further studied functional pathway of FHL1.
In cell lines, this paper found top rank of differences pathways (PI3K-Akt signaling pathway and JAK-STAT signaling pathway), which might be connected with drug resistance (Fig. 5c), and FHL1 was involved in JAK-STAT signaling pathway (CCND1, FHL1, IL2RA, IL6ST, AKT3) (Fig. 5d), which had shown promising prognostic values. In KEGG, surprise was that cell signaling pathways of FHL1 expression were involved in “PI3K-Akt signaling pathway” and “MAPK signaling pathway”, both of which were included in “JAK-STAT signaling pathways” (Fig. 5d). Gene CCND1 played a vital role in the regulation of cell proliferation and hematopoietic differentiation [18, 30]. AKT3 control over cell proliferation and cell viability was manifold [20]. In the JAK-STAT pathway, following the binding of cytokines to their cognate receptor, STATs were enabled to modulate the expression of target genes by members of the JAK family of tyrosine kinases. In addition to the activation of STATs, JAKs mediate the recruitment of other molecules such as the MAP kinases, PI3 kinase etc (PI3K-Akt signaling pathway and MAPK signaling pathway existed in the JAK-STAT pathway). These molecules process downstream signals via the Ras-Raf-MAP kinase and PI3 kinase pathways, led to the activation of additional transcription factors. PI3K-Akt-mTOR pathway amplification was associated with reduced OS and DFS [20]. So we speculated that PI3K-Akt signaling pathway and MAPK signaling pathway played a pivotal role in JAK-STAT signaling pathway. In platelet activation pathway and another cancer pathway (VEGF signaling pathway also was found, including PI3K-Akt signaling pathway and MAPK signaling pathway) (Fig. 4d), FHL1 played a driver role in the aggressiveness of cancer cells and a potential catalyzes role in tumor angiogenesis. In such a scenario, gene mutations would cooperate with the genes and protein expression profile to present a cellular phenotype that depended on both components. In the present study, FHL1 as a target gene of downstream of TLX1 also acted as anti-oncogene was a dual function regulator, serving as either an activator or a repressor depending on cell and promoter context [31]. Together with these genes and pathways, FHL1 was the key gene in signaling pathway, especially in PI3K-Akt signaling pathway and JAK-STAT signaling pathway and might play a crucial role in the cancer process. Gene knockout and pathological experiments may also be further studied to determine the function of FHL1. Our results proved FHL1 as a potential prognosticator and therapeutic target for IP-AML and provided new perspectives into the pathogenesis and precision medicine of IP-AML.