Variety of combinations of genetic abnormalities like chromosomal translocations, gene mutations, and epigenetic modifications could result in the occurrence of unique form of acute myeloid leukemia (AML)12. An integrated mutational profiling of AML cases could affect treatment response and high rate of relapse or resistance after chemotherapy with DNA-targeting drugs13,14. Interpretation of the role of differentially expressed genes (DEGs) and potential pathways in AML drug resistance is very complex. This is because genes are frequently co-occurring in numerous cross-talking cascades, involving in various pathways and regulating different overlapped biological process depending on the type of the cells and cancers15.
It could be hypothesized that multiple subgroups of AMLs with different genetic background which are under treatment of various regimens might have similar regulatory gene(s) or pathway(s) related to their chemo-resistance phenotype. In this study we wished to investigate deregulated genes and enriched pathways involved in drug resistance in AML patients under treatment of DNA-damaging agents including Anthracyclines, Cytarabine and Gemtuzumab ozogamicin. Identification of overlapping biological processes which contribute to chemo-refractory relapse in patients with AML following chemotherapy could open a new insight into the finding of novel molecular targets.
Given the above hypothesis, analysis of the transcriptomic profiles of AML samples (patients, leukemic stem cells and cell lines) was performed on two groups, chemo-resistance against chemo-sensitive. Moreover, a comparative meta-analysis in three ways was conducted based on sample type (only for patient’s sample), age (only adult; due to unavailability of sensitive sample in childhood group) and samples in total (with any criteria). The data resulted in the identification of 34 common DEGs that were statistically correlated with AML chemo-resistance. In the next step, gene set enrichment analysis using DAVID/Enrichr/Cytoscape (stringApp) was done to identify possible signaling pathways which were enriched among candidate DEGS, and could be associated with chemo-refractory relapse to DNA-damaging compounds in AML patients after chemotherapy.
Our data revealed the involvement of five major signaling pathways associated with chemo-refractory relapse in AML samples. These signaling pathways were as follow: Neurotrophin, Estrogen, cAMP, Toll-like receptor and Renal cell carcinoma. Among the above pathways, Neurotrophin signaling pathway was found to be the most significantly over-represented signal transduction for driving drug resistance in AML as indicated in Table 1–3. This biological signaling pathway is highlighted in nervous system for neuronal growth, differentiation, survival, and plasticity16. Its tyrosine kinase receptors (tropomyosin receptor kinase (Trk)) including Trk A, B and C, express in a variety of human tissues and support cell survival in multiple solid and liquid tumors17,18. Moreover, Neurotrophin/Trk signaling pathway has been found connected with a variety of intracellular cascades including MAPK pathway, PI3k/AKT pathway, and PLC pathway, providing growth and survival advantage for cells19,20. On the contrary, the p75 neurotrophin receptor (p75NTR), another receptor of neurotrophins, induces the expression of pro-apoptotic genes through activation of p38 and JNK pathways20.
Li and colleagues showed that the activation of neurotrophin/Trk pathway could result in leukemogenesis as well as survival of leukemic cells and poor outcome after chemotherapy21. Furthermore, Ho and co-workers reported a similar biological behavior of cell survival as well as chemo-resistance phenotype in neuroblastoma cells when exposed to DNA-damaging compounds22. Their data suggested that TrkB-BDNF pathway plays a key role in resistance to chemotherapy by activation of PI3K/AKT survival pathway as a downstream mediator.
Our meta-analysis data revealed that the expression of AKT3, RPS6KA2, c-Jun, SORT1 and MATK genes was significantly altered in chemo-resistance AML samples. SORT1, AKT3 and RPS6KA2 genes were up-regulated but c-Jun and MATK were downregulated in AML chemo-resistance group compared to the -sensitive group (see Fig. 6B). It has been reported that AKT3 has an important role in DNA double strand break repair and chemotherapeutic resistance23. It has been documented that both AKT3 and RPS6KA2 genes could act in parallel as the mediators of PI3k/AKT and MAPK pathway under the impression of neurotrophin signals17,24. c-Jun has been reported to play an apoptotic role in neurotrophin/Trk signaling pathway20.
SORT1 (also known as Sortilin 1) acts in a Trk-dependent manner in neurotrophin signaling pathway and its overexpression has been reported in human cancer cells20,25. The contribution of SORT1 with Trk receptors promotes cell survival and characterized as a oncogenic factor for cells20. This gene was the second significant up-regulated gene in chemo-resistant AML samples, which may induce resistance to chemotherapy through neurotrophin signaling pathway. It was reported that the expression of SOTR1 was elevated in adult Acute B Lymphoblastic Leukemia (B-ALL) cases after chemotherapy, which was correlated to relapse and/or B-ALL-related death26. Recent reports have pointed to the clinical application of neurotrophic tyrosine receptor kinase (NTRK) inhibitors as chemotherapy agents for treatment of hematological malignancy27. However, in view of our meta-analysis data, it seems that the application of Trk inhibitors alone may not be affective, because other functional genes/mediators in this pathway may interfere and strength this pathway resulting in cell survival.
cAMP signaling pathway is the second pathway that was identified in our chemo-resistance AML samples. This pathway is one of the important cascades associated with anthracycline resistance in AML patients28. It has been demonstrated that cAMP plays a crucial role in the reduction of response to DNA-damaging reagents in CML cells28. Moreover, it has been shown that cAMP signaling pathway is under regulation of G protein-coupled receptors (GPCRs) which contribute to the development of AML 29. It has been reported that elevation of cAMP signaling suppresses apoptosis-induced JNK activation30.
Our meta-analysis data showed that GABBR1, a member of GPCR family, had an increased expression in chemo-resistance samples compared to the sensitive ones (logFC 1.6) (Fig. 6b)31. GABBR1 has been introduced as a survival associated marker for AML32. Excessive signal transduction through GABBR1 triggers growth and migration of cancer cells32. These findings support our data showing the association of increased expression of GABBR1 and chemoresistance in AML samples.
In addition to GABBR1, the expression level of other cAMP-related genes including ARAP3, AKT3 were increased, but c-Jun was down regulated. ARAP3 is a GTPase regulatory protein, which its up-regulation as a co-regulator with NEDD9 could significantly mediate progression of breast cancer cells and increase likelihood of metastatic relapse of tumor cells33. ARAP3 and AKT3 are two down-stream elements of GPCRs oncogenic pathway. The critical role of ARAP3 and AKT isoforms was shown in regulating the developmental angiogenesis. These two proteins are common substrate for PI3K pathways which play an essential role in angiogenesis34,35. The importance of angiogenesis in AML as a source of drug resistance and relapse was documented in several clinical studies36,37.
Given the highest expression of ARAP3 gene among 34 DEGs in our chemoresistance samples, it could be suggested that increased level of ARAP3 may correlate with increased angiogenesis through PI3K pathway. This could also provide an explanation for the development of chemoresistance response of AML patients to treatment. Therefore, ARAP3 gene could be introduced as a high-risk marker in AML relapse, and could be considered as a new target for AML therapy.
Besides, many reports have confirmed the important role of Akt-related pathway in the development of resistance against DNA damaging drugs in tumor cells11,22,23,38,39. It has been shown that Akt-related pathway could lead to resume DNA replication by recovery of genome stability, and drive cancer cells to M phase through stimulate expression of CDKN1A (cyclin-dependent kinase inhibitor 1A). Moreover, it has been shown that the increased AKT3 gene expression could promote tumor malignancy and resistance to DNA damaging chemotherapy compounds through activation of DNA repair pathway in glioma tumor cells23,40.
The next signaling pathway which was deregulated in our chemoresistance AML samples was the estrogen signaling pathway. In this pathway, AKT3, c-Jun and GABBR1 genes were deregulated in chemoresistance AML samples, of which AKT3 and GABBR1 were upregulated, but c-Jun was down regulated. Several studies have characterized the importance of estrogen receptors and their contribution in the response of AML cells to chemotherapy41–44. Recently, a preclinical study has considered estrogen receptors as a potential target to enhance chemotherapy for patients with AML 41. Given the increased expression of AKT3 and GABBR1 in chemoresistance AML samples, as shown in our meta-analysis data, it could be suggested that the estrogen signaling pathway might play an important role in the protection of leukemic cells from apoptosis.
Moreover, it has been reported that estrogen signaling pathway was altered in breast cancer cells in response to Anthracyclines45. This finding could support our data on the effects of estrogen signaling pathway in the development of chemoresistance in AML.
Toll like receptor (TLR) signaling pathway was the last signaling pathway in our gene set enrichment analysis data, which was correlated with chemoresistance behavior of AML cells. These receptors are pattern recognition receptors (PRRs) which are expressed in hematopoietic stem and progenitor cells (HSPCs), effector immune cells, non-immune cells like stromal cells and also AML cells46–48. Moreover, these receptors conduct downstream intracellular signaling cascades upon two distinct adaptor proteins: MyD88 and TRIF. TLR family provides cyto-protective response after activation by recognizing exogenous ligands including various pathogen/microbial components (PAMPs; pathogen-associated molecular patterns) or endogenous ligands (DAMPs, damage-associated molecular patterns). Activation of TLR family lead to cell stress such as DNA damaging, reactive oxygen species (ROS), and heat shock proteins (Hsps) and has a considerable function in inflammatory response through downstream pathways nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB), AP-1, and p38 MAPK pathways46,49.
Anthracyclines, as the immune-stimulatory chemotherapeutic agent, can promote TLRs-mediated immunogenic apoptotic cell death through increased emission of DAMPs by damaging DNA in tumor cells50. Based on our GSEA results, differential expression of several genes including PELI2 (Pellino2), RPS6KA2(RSK3), S100B, c-Jun, AKT3, and SPP1, may contribute to the aberrant signal transduction of TLRs upon TRIF/MyD88- mediated induction signaling in chemoresistance AML group.
Our data showed an enhanced expression of PELI2 gene in chemoresistance group. PELI2 encodes one of the members of the E3 ubiquitin ligases which regulate activation of NFκB and MAPK cascades downstream of TLR signaling pathway. Studies on PELI2 have shown a reciprocal regulating interplay between PELI2 and IRAK151–53. PELI2 interacts with IRAK1 and can be a kinase substrate of IRAK154. In addition, it efficiently mediates polyubiquitination of IRAK1 in both Lys-63 and Lys-48 and induces TAK1-dependent JNK and ERK activation52–54. However, It appears that PELI2 involves in various cascades with a cell-type specific manner55. Recently, it was reported that PELI2 has a positive role in regulation of signaling-mediated NLRP3 inflammasome and increase caspase1-mediated activation of two immunoregulatory cytokines from IL-1 family, IL-1β and IL-18, in post-translational stage. PELI2 activates inflammasome complex through concurrent of ubiquitination of two parallel targets, NLRP3 and IRAK1. The inactivation of IRAK1 could suppress the activation of inflammasome52.
Based on observations that IL-1β and IL-18 can contribute to AML anti-cancer drug resistance, and based on our data showing enhanced expression of PELI2 gene, mediating IL-1β and IL-18 activation may be a distinct plausible important mechanism by which PELI2 involved in emergence of drug resistance56–59. In spite of this, some studies highlighted the central role of dysregulated IRAK1 and IRAK4 signaling in chemotherapy resistance60. Understanding the relationship between deregulated expression of PELI2 gene and AML chemotherapy failure remains a challenge and further in vitro studies can provide important clues for its potential therapeutic usefulness.
It may be helpful to co-treatment of AML chemo-resistant cells with the PELI2 inhibitor plus TLR agonist to synergistically activate immune system and inhibit cell proliferation.
The second gene in TLR signaling pathway was RPS6KA2 (RSK3) which belongs to the ribosomal S6 kinase family. It has been reported that the up-regulation of RPS6KA2 gene could mediate drug-resistant phenotype in pancreatic and breast cancer27,61,62. More recently, it has been reported that RSK inhibition leads to overcoming vinca alkaloid resistance by inhibiting AML cell proliferation and inducing apoptosis63,64. In contrast, our data showed a reduced expression of RPS6KA2 in chemoresistant AML group. A previous study reported that RPS6KA2 was activated in vitro by c-Jun N-terminal kinase (JNK) and might be one of the downstream effectors of JNK in vivo in skeletal muscles contributed to glycogen synthase by insulin64. Our KEGG data showed the involvement of down regulation of RPS6KA2 in insulin resistance.
JNK signaling pathway has been shown to be activated by multiple receptors including GPCRs, TLRs, neurotrophin receptors (Trks), and estrogen receptors (ER) which generally promotes cell death and apoptosis through activation of c-Jun, an important pro-apoptotic protein, and inhibition of Akt-inducing survival signaling65–68. Interestingly, as illustrated in Fig. 8, consistent with the above facts, our meta- and gene set enrichment analysis in AML chemoresistance samples showed significant inhibition of JNK signaling due to simultaneous down-regulation of c-Jun and up-regulation of AKT3 expression.
Several studies have highlighted the critical role of JNK in Anthracycline induced apoptosis in AML cells69,70. These studies hypothesized that failure in JNK activation could be one of the main cause of resistance of AML cells to Anthracycline-containing treatment protocols69,70. These reports can further support our in silico findings that overexpression of AKT3 and down-regulation of c-Jun could function as one of the main molecular mechanism for resistance of AML patients to chemotherapeutic protocols. Moreover, recently the importance of Akt inhibitors to improve the efficacy of DNA-targeting drugs has been suggested38. Therefore, we can suggest that reduced impact of JNK signaling at the intersection between multiple signaling pathways in AML cell may strengthen survival signaling in these cells against DNA-damaging drugs, resulting in chemoresistance phenotype.