The Emerging Role of Pyruvate Kinase M2 in Regulating Glutaminolysis via c-Myc

Cancer cells reprogram their nutrition metabolism to meet their high bioenergetic and biosynthetic demands in support of rapid growth and continuous proliferation [1]. Both glycolysis and glutaminolysis are altered in cancer cells [2]. Glycolysis is a universal pathway used by all living cells for energy production, which can occur in both aerobic and anaerobic states. However, about a century ago, Otto Warburg first discovered that the glycolysis rate is ~200 times higher in cancer cells compared to healthy cells and cancer cells predominantly undergo anaerobic glycolysis regardless of whether oxygen is present [2,3]. This phenomenon is later termed Warburg effect. After its discovery, scientists have come up with many theories for why cancer cells switch to inefficient anaerobic glycolysis. One widely accepted theory is that Warburg effect allows cancer cells to maintain large pools of intermediates which provide building blocks for synthesis of nucleotides, lipids and amino acid, assisting the rapid proliferation and growth of cancer cells [4].

Cancer cells reprogram their nutrition metabolism to meet their high bioenergetic and biosynthetic demands in support of rapid growth and continuous proliferation [1]. Both glycolysis and glutaminolysis are altered in cancer cells [2]. Glycolysis is a universal pathway used by all living cells for energy production, which can occur in both aerobic and anaerobic states. However, about a century ago, Otto Warburg first discovered that the glycolysis rate is ~200 times higher in cancer cells compared to healthy cells and cancer cells predominantly undergo anaerobic glycolysis regardless of whether oxygen is present [2,3]. This phenomenon is later termed Warburg effect. After its discovery, scientists have come up with many theories for why cancer cells switch to inefficient anaerobic glycolysis. One widely accepted theory is that Warburg effect allows cancer cells to maintain large pools of intermediates which provide building blocks for synthesis of nucleotides, lipids and amino acid, assisting the rapid proliferation and growth of cancer cells [4].
Warburg effect limits entry of pyruvate into TCA cycle in cancer cells. To compensate for the reduction in carbon coming from glycolysis, cancer cells switch to the glutaminolysis which utilizes glutamine to replenish the TCA cycle [5]. In addition, glutaminolysis also provides reducing power by converting glutamate to glutathione (GSH), the most abundant anti-oxidant in mammalian cells in handling oxidative stress [5]. Elevated glutaminolysis has been recognized as a critical hallmark of cancer [6]. Not only that cancer patients have much lower concentrations of glutamine in their blood circulation compared to healthy individual [6], but also, the reduction in glutamine consumption can restrain the cancer progression to some extent [7]. Glutamine, the most abundant amino acid in blood circulation, is beneficial for cancer cells as it supplies more carbon and nitrogen for macromolecule biosynthesis and maintenance of redox balance [8][9][10]. By this means, with increased glutamine being converted via glutaminolysis, cancer cells display enhanced cell proliferation rate and reduced cell death [11].
Arch Cancer Biol Ther. 2021 Volume 2, Issue 1 20 between glycolysis and glutaminolysis in cancer cells" [12], the authors demonstrated that the pyruvate kinase M2 (PKM2), a critical enzyme in glycolysis, helps to switch the glutaminolysis pathway in cancer cells through internal ribosome entry site (IRES)-dependent translation of c-Myc. This study uncovers an important mechanism how cancer cells coordinate glycolysis and glutaminolysis and also sheds light on the novel role of PKM2 as a regulator of glutaminolysis. Nevertheless, the unique way of PKM2 in regulation of glutaminolysis in cancer cells also brings up some intriguing questions which are worthy of further investigations ( Figure 1): Does PKM2 manipulate any other gene expression through IRES-dependent translation? Does PKM2 regulates glutaminolysis in other cell types besides cancer cells? What is the exact role of PKM2 in IRES-dependent translation of c-Myc?

The Unique Role of PKM2 in Regulation of Glutaminolysis
Pyruvate kinase is a key enzyme in glycolysis, which catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate at the last, yet a rate-limiting, step of glycolysis. Although there are several different isoforms of pyruvate kinase, cancer cells highly express M2 isoform [13]. More interestingly, PKM2 in cancer cells is predominantly enzymatic inactive form, dimer PKM2 [14]. Compared to its active tetramer PKM2, the low-activity-dimer PKM2 has compromised affinity for its substrate PEP, leading to accumulation of glycolytic intermediates. Despite its glycolytic roles, PKM2 in cancer cells have additional non-glycolytic functions [14,15]. For instance, PKM2 in cancer cells is identified to regulate HIF1α activity as a transcriptional activator [16]; and PKM2 is also demonstrated to phosphorylate STAT3 using PEP as a phosphate donor in cancer cells [17]. These various noncanonical functions suggest that roles of PKM2 in cancer cells are complicated, and there might be other nonglycolytic functions which have not been fully elucidated. Here in the new study "Pyruvate kinase M2 coordinates metabolism switch between glycolysis and glutaminolysis in cancer cells", the authors uncovered another novel nonglycolytic function of PKM2 in cancer cells.
In recent study, Li and co-workers demonstrated the mechanism how PKM2 coordinates the glycolysis and glutaminolysis pathways. The authors showed for the first time a crucial role of inactive dimer PKM2 in regulating

IRES-dependent protein translation in cancer cells.
This study demonstrated that PKM2 dimer, promoted by growth stimulations, elevates glutaminolysis by upregulating mitochondrial glutaminase I (GLS-1) via interacting with c-myc IRES-RNA and facilitating IRES-dependent translation of c-Myc. The traditional understanding of the role of PKM2 in cancer metabolism is that the inactive dimer PKM2 support cancer cell growth by allowing the glycolytic intermediates to build-up and re-direct into anabolic pathways [18], an advance made by the latest report is that dimer PKM2 can also modulate the augmentation of glutaminolysis, feeding bio-synthesis materials to cancer cells.

Could Dimeric PKM2 Regulate IRESdependent Translation of Other Proteins?
A number of proliferation and survival related genes are regulated at translational level [19]. This is important to cancer cells since translational regulation provides them rapid adjustment to stressful environment [20]. Accumulating evidence shows that IRES-dependent translation is important for many cancer-related proteins [21][22][23], suggesting that IRES-mediated translation is vital for cancer cells. Our work showed that PKM2 can regulate GLS-1 through IRES-dependent translation of c-Myc. Since c-Myc is an important regulator in cancer cells [24], PKM2 might regulate other c-Myc related pathways such as cell cycle, apoptosis and proliferation [25][26][27][28], which are worth further investigations.
In addition to c-Myc, many other cancer-progression associated genes have been reported to be regulated by IRES-mediated translation, such as VEGF, IGF1R, cyclin D1 and XIAP [29][30][31][32][33], Furthermore, IRES-mediated translation of these genes have been found critical for many processes of cancer cells including metastasis and angiogenesis [31,34,35]. Interestingly, dimer PKM2 is found to promote cancer cell metastasis and angiogenesis [36,37]. It is plausible that the dimer PKM2 might be able to regulate these genes through IRES-dependent translation. It is worthy to further test if PKM2 plays a role in regulation of these cancer promoting genes through IRES-dependent translation in cancer cells, which may suggest a broader implication of dimer PKM2 in reprogramming and coordinating multiple cellular processes in cancer cells.

Could
Dimeric PKM2 Regulate Glutaminolysis in Non-cancer Cell Types?
Besides tumor cells, metabolic reprogramming of glutaminolysis is observed in multiple cell types such as immune cells [38][39][40]. The role of dimer PKM2 in regulation of glutaminolysis in non-cancer cell types merits further investigations.
Macrophages and neutrophils stimulated by LPS or bacterial infection utilize glutaminolysis to sustain ROS production by generating NADH [41]. Furthermore, macrophages and neutrophils upon microbial activation display an increased level of PKM2 expression [42,43] and PKM2 in LPS-activated macrophages are primarily dimers [43]. Altogether, innate immune cells such as macrophages following microbial challenge might elevate dimer PKM2 to rewire glutaminolysis through a similar mechanism as was observed in cancer cells. Noticeably, the role of dimer PKM2 in macrophages might not act through the regulation of IRES-dependent translation of c-Myc as c-Myc can drive macrophages to alternative activated phenotype [44] which mainly have tetramer PKM2 [43]. Nevertheless, it is still worthy to investigate whether dimer PKM2 can regulate the glutaminolysis in innate immune cells and the underneath mechanisms if it can.
Besides the innate immune cells, it is well appreciated that dimer PKM2 is essential for CD4 + T cell activation [45]. Moreover, CD4 + T cell activation also relies on glutaminolysis and c-Myc is an indispensable factor in this process [46][47][48]. So, it is possible that dimer PKM2 mediates glutaminolysis activation in the process of T cell activation via regulating c-Myc by IRES-dependent translation.
In addition to immune cells, emerging evidence suggests that glutaminolysis is required for the activation of myofibroblasts [49,50], whose persistence is a hallmark of fibrotic diseases of multiply tissues and organs [50]. PKM2 is expressed in fibroblasts. Thus, it is interesting and important to analyze potential role of dimer PKM2 in gluaminolysis regulation in fibroblasts differentiation and activation to myofibroblasts, which may shed light on the molecular mechanism of organ and tissue fibrosis development and progression.

What are the Possible Roles of PKM2 in IRES-dependent Translation of C-Myc?
Dimer PKM2 regulates c-Myc expression via the IRESdependent translation. However, the detailed mechanism about how PKM2 regulates c-Myc expression via IRESdependent translation remains unclear. IRES-dependent translation involves multiple different proteins [22,51]. Inferring from the observations, it is plausible that PKM2 may facilitate assembly of hnRNP L/K into c-myc IRES complex. However, further study is needed to elucidate how PKM2 regulates the assembly process of c-myc IRES complex. In addition, it is also unknown whether dimer PKM2 is directly involved in the IRES-dependent c-Myc translation. Since the recruitment of translation initiation factors to IRES sequence and structure is critical for IRES-dependent translation [52,53]. It is reasonable to