Targeted Depletion of Amino Acids As a Novel Therapy for Acute Leukemia and Other Cancers: Mechanisms and Countermechanisms

Introduction:

Metabolic reprogramming by cancer cells to allow proliferation and survival suggests targeting of relatively cancer cell-specific metabolic processes as a potential cancer therapy. The amino acid (aa) glutamine (GLN) functions as an exchange factor to facilitate cell import of essential amino acids (EAA), which positively regulate translation by the mTORC1 pathway (via phosphorylation of S70K and 4EBP1), allowing proliferation. Most cancer cells also rely on GLN, rather than glucose for citric acid cycle (TCA) anaplerosis, and as a source of energy, anti-oxidants and components for protein synthesis.

L-asparaginase (L-Ase), an enzyme that breaks down extracellular asparagine (ASN, the least prevalent intracellular aa), is used in the treatment of ALL. L-Ase is also glutaminolytic, resulting in GLN depletion and apoptosis that is suppressed by ASN repletion, which modulates the cell stress responses (ISR, upregulatingATF4, CHOP, aa transporters, and asparagine synthetase (ASNS)). Thus, (i) ASN is a critical signal preventing cell death from GLN depletion; (ii) ASN repletion (via ASNS) may be the important function of GLN within cancer cells, and (iii) mechanisms that deplete bothkey aa may be synergistic in implementing cancer cell death

Apart from non-EAA synthesis and aa uptake (#1 in Fig. 1A), there are two major pathways of cellular aa repletion: (i) autophagy, a process whereby damaged proteins are delivered to the lysosome for degradation (#2), and (ii) the ubiquitin-proteasome system (UPS, #3), which also degrades damaged or misfolded cell proteins, allowing aa recycling. Notably, UPS inhibition significantly decreases ASN (andcystine) levels.

The aim of our studies is to explore mechanisms of depleting intracellular GLN and ASN levels in cancer cells, firstinvestigating the potential synergistic effects of combining L-Ase, with Chloroquine (CQ, autophagy inhibition) and Bortezomib (BTZ, proteasome inhibition), and then analyzing cancer cell counter mechanisms.

Results:

We performed kill-curves with individual drugs, and then combinations of L-ase, CQ and BTZ in REH (ALL) cells. Notably, inhibitory effects on aarepletion pathways, as determined by western blot analysis of cell lysates at 12h (Fig. 1B), were seen with a combination of significantly lowered doses of each drug [BTZ 2nM (40% of LD₅₀); L-Ase 0.2IU (15%); CQ 100mM (50%)]. The mTORC1 pathway is especially susceptible to inhibition by drug combination-mediated aa depletion (decreased phosphorylation of 4EBP1 and S6K1; compare lanes 2-4 & 5-8), while autophagy (monitored by increasing levels of LC3-II) is also inhibited. Cell viability was assessed after 48h. Although the low doses of each drug used has a minimal impact on viability (range 75-130% of control), the combination above (2nM;0.2IU;100mM) results in synergistic cell death [55% (n = 1)]. We will examine further the effects of this drug combination on normal CD34+ cells, prior to studies of efficacy inxeno-transplant models.

Most tumors are metabolically flexible, e.g., they can use glucose if deprived of GLN to replenish TCA, and, via TCA intermediates, increase GLN levels, and thereby ASN, via pyruvate carboxylase (PC), transaminases (GOT1, 2), glutaminesynthetases(GDH, GS) and ASNS (see Fig. 1 pathways). Thus, we interrogated, byqPCR, potentially relevant pathways that may be used to evade glutamine and asparagine depletion-induced apoptosis (Fig. 1C). Of 12 genes tested, GLN deprivation significantlyupregulatesGLS1, GOT1, and ASNS to increase ASN levels, while the ISR is activated (CHOP), and SLC7A11, a cysteine importer upregulated in tumors (for glutathione production) is also significantly upregulated. Preliminary studies of REH and A549 (lung cancer) cells suggest a common theme in metabolic responses to GLN depletion in diverse cancer cells is ASN synthesis through GOT1 and ASNS upregulation, and likely ROS production throughcystineuptake.

Conclusions: Commonly, inhibition of one metabolic pathway results in upregulation of another. Our studies indicate that combination therapy, using low doses of available, well-studied drugs depletes keyaa ASN and GLN, and prevents their repletion, causing cancer cell death. In addition, our studies of the cellular responses to GLN depletion alone indicate additional targets that should be considered to prevent ASN-mediated inhibition of cell death in diverse cancer types.

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Figure 1

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