Nicotinamide riboside (NR) supplementation helps TILs overcome the mitochondrial dysfunction and preserves their ability to attack tumors

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A Ludwig Cancer Research study has uncovered a mechanism by which the tumor’s harsh internal environment sabotages T lymphocytes, leading cellular agents of the anticancer immune response.

Reported in Nature Immunology, the study describes how a variety of stressors prevalent in the tumor microenvironment disrupt the power generators, or mitochondria, of tumor-infiltrating T lymphocytes (TILs), pushing them into a permanently sluggish state known as terminal exhaustion.

The study, led by Ludwig Lausanne Associate Member Ping-Chih Ho, also found that a widely available nutritional supplement—nicotinamide riboside (NR) – helps TILs overcome the mitochondrial dysfunction and preserves their ability to attack tumors in mouse models of melanoma and colon cancer.

“TILs often have a high affinity for antigens expressed by cancer cells,” says Ho.

“This means that, in principle, they should attack cancer cells vigorously. But we often don’t see that. People have always wondered why because it suggests that the best soldiers of the immune system are vulnerable when they enter the battlefield of the tumor.

Our study provides a mechanistic understanding of why this happens and suggests a possible strategy for preventing the effect that can be quickly evaluated in clinical trials.”

The inner recesses of tumors are often starved of oxygen and essential nutrients, such as the sugar glucose. Cells in these stressful conditions adjust their metabolic processes to compensate – for example, by making more mitochondria and burning their fat reserves for energy.

In tumors, prolonged stimulation by cancer antigens is known to push TILs into an exhausted state marked by the expression of PD-1 – a signaling protein that suppresses T cell responses and is targeted by existing “checkpoint blockade” immunotherapies.

If sustained, such exhaustion can become permanent, persisting even when the stimulus of cancer antigens is removed.

Ho and his colleagues found that exhausted TILs are packed with damaged – or ‘depolarized’ – mitochondria. Like old batteries, depolarized mitochondria essentially lack the voltage the organelles require to generate energy.

“Our functional analysis revealed that those T cells with the most depolarized mitochondria behaved most like terminally exhausted T cells,” said Ho.

Ho and colleagues show that the accumulation of depolarized mitochondria is caused primarily by the TIL’s inability to remove and digest damaged ones through a process known as mitophagy.

“The TILs can still make new mitochondria but, because they don’t remove the old ones, they lack the space to accommodate the new ones,” said Ho.

The genomes of these TILs are also reprogrammed by epigenetic modifications – chemical groups added to DNA and its protein packaging – to induce patterns of gene expression associated with terminal exhaustion.

The researchers found that the breakdown in mitophagy stems from a convergence of factors: chronic stimulation by cancer antigens, PD-1 signaling and the metabolic stress of nutrient and oxygen deprivation.

They also show that the epigenetic reprograming that fixes TILs in a terminally exhausted state is a consequence, not a cause, of the mitochondrial dysfunction.

Related work done by other researchers – including co-authors in the current study, Ludwig Lausanne Investigator Nicola Vannini and Ludwig Lausanne Branch Director George Coukos – has shown that NR, a chemical analogue of vitamin B3, can boost mitophagy and improve mitochondrial fitness in a variety of other cell types.

With this in mind, the researchers explored whether NR might also prevent TILs from committing to terminal exhaustion.

Their cell culture experiments showed that the supplement improved the mitochondrial fitness and function of T cells grown under stressors resembling those of the tumor microenvironment.

More notably, dietary supplementation with NR stimulated the anti-tumor activity of TILs in a mouse model of skin cancer and colon cancer. When combined with anti-PD-1 and another type of checkpoint blockade, anti-CTLA-4 immunotherapy, it significantly inhibited the growth of tumors in the mice.

“We have shown that we may be able to use a nutritional approach to improve checkpoint blockade immunotherapy for cancer,” said Ho.

He and his colleagues are now exploring the signals from depolarized mitochondria that epigenetically reprogram TILs for terminal exhaustion—information that could be more generally applied to improve cancer immunotherapy.


Rapidly proliferating cancer cells have altered metabolic needs, including an increased rate of nicotinamide adenine dinucleotide (NAD) cycling relative to normal cells1–3. NAD is an essential substrate for maintaining cellular bioenergetics and supporting NAD-dependent proteins integral to DNA repair, genomic integrity, and regulation of transcription, signaling, and oxidative stress3–5.

In several cancer types, sustained depletion of NAD has been shown to trigger apoptosis and autophagy, indicating cellular dependence on maintenance of adequate levels6–8.

Cellular NAD can be produced through several redundant synthesis pathways, some of which include enzymes that are over-expressed or silenced in certain cancers3,9–16. The salvage pathway represents one such pathway of key importance in cancer, functioning to recycle nicotinamide (NAM), the product of NAD+-consuming enzymes, back into NAD+17.

In the salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT) acts as the rate-limiting enzyme and produces nicotinamide mononucleotide (NMN), an NAD precursor3,15–17. In certain cancers, NAMPT expression has been shown to promote carcinogenesis and is associated with worse prognosis3,9,16.

Preclinically, pharmacological inhibitors of NAMPT have been shown to deplete NAD, resulting in loss of cell viability in a variety of cancer types6–8,10,18–21. Because the cellular functions of NAD are broad, NAMPT inhibitors (NAMPTis) may have multiple anticancer effects including inhibition of energy metabolism, susceptibility to oxidative stress, and impairment of DNA damage repair2,9,21–23. NAMPT is currently the only NAD+ production enzyme that has been targeted in the clinic2,5,24.

First-generation NAMPTis were tested in several early phase clinical trials in unselected adult patients with advanced cancers, yielding a disease control rate of about 25% but few objective responses25–30. Bone marrow suppression, especially thrombocytopenia, was dose-limiting in these trials, as were gastrointestinal side-effects25–30.

In large animal studies, retinal and cardiac toxicities were observed, although these were not reported in human patients31,32. Given the paucity of objective responses and concerns about NAMPTi-associated toxicities, development of this class of agents was halted33.

OT-82 (OncoTartis) is a novel, oral, small molecule inhibitor of NAMPT currently undergoing clinical assessment for hematological malignancies. While initially discovered using an assay for hematopoietic tissue-specific cytotoxic agents, its mechanism was revealed to be a NAMPTi.

Early data suggest that OT-82 possesses a more favorable toxicity profile than earlier-generation NAMPTis, particularly with regard to retinal and cardiac toxicities that were observed in animal studies of earlier-generation molecules but were not observed with OT-8234.

In addition, recent evidence has emerged demonstrating that certain tumor types may be more sensitive to inhibition of NAMPT due to differential vulnerabilities in NAD-related processes9. Ewing sarcoma (EWS), a pediatric bone and soft tissue cancer, represents one such malignancy as recent studies have revealed the presence of intrinsic defects in DNA damage repair and metabolic reprogramming35–39.

Furthermore, in vitro data using early-generation NAMPTis suggests that EWS cells may be more sensitive than other cancer cell types40,41. However, since EWS patients were never treated in any early NAMPTi clinical trials, the potential clinical efficacy of this class of agents remains untested in this population. Thus, the purpose of this study was to evaluate the activity and mechanistic effects of the latest-generation NAMPT inhibitor OT-82 in in vitro and in vivo models of EWS.

Discussion

In this study we show that loss of NAMPT activity through genetic or pharmacological inhibition results in impaired EWS cell proliferation and survival, demonstrating the dependency of EWS on NAMPT. We provide the first evaluation in solid tumor models of the activity of OT-82, a recently described, novel NAMPTi that was selected for clinical development due to specific cytotoxicity against hematologic malignancies34.

Remarkably, we observed equal or lower IC50 values for OT-82 in our panel of EWS cell lines than those reported for the preclinical models of hematologic malignancies, suggesting that EWS may represent a selectively sensitive candidate solid tumor34,58.

The enhanced sensitivity of EWS to NAMPT inhibition may be, in part, related to the known homologous repair deficiency that is a feature of EWS35, as DNA repair deficiencies have been linked to NAMPTi sensitivity in other solid tumor types23 and may increase dependence on the NAD-dependent PARPs59,60.

Other factors specific to EWS may also contribute to NAMPTi sensitivity, including the presence of the oncogenic fusion protein EWS-FLI140, which has been shown to drive reprogramming of NAD-dependent metabolic enzymes in EWS, such as PHGDH and LDHA37–39,44, and reliance on other NAD-dependent proteins such as SIRT161,62.

Our mechanistic studies of OT-82 in EWS indicate that it functions by depleting cellular NAD, which in turn results in both G2 arrest and apoptosis. This is partially consistent with its effect in leukemia models, where OT-82 was observed to induce apoptosis, but not cell-cycle arrest34,58.

Interestingly, use of the earlier-generation NAMPTi FK-866 has been associated with G2/M arrest in models of other solid tumors63, suggesting that the specific mechanistic effects of NAMPT inhibition may be cell-type specific. The effect of OT-82 on ROS induction in our models represents an additional example of the differential effects NAMPTis may have on different cell types.

Induction of ROS is a known mechanism of NAMPTi-induced killing in a range of cancer cell types45–50. However, as we demonstrated in this study, induction of ROS was not responsible for OT-82-induced cell death in EWS, suggesting that ROS production is a byproduct of OT-82-induced cell death.

This is in contrast to the effect of OT-82-induced ROS reported in PDX models of pediatric acute lymphoblastic leukemia58 but is concordant with prior results using the NAMPTi GNE-618 in EWS41, and suggests that the differential effects are due to the affected cell type, as opposed to the particular NAMPTi used.

In vivo, OT-82 treatment resulted in substantial inhibition of EWS xenograft tumor growth and prolonged survival at doses demonstrating pharmacodynamic evidence of on-target activity in tumors, using NAD concentration and PARP activity. Notably, PARP activity assays have been successfully used in the clinic64 and therefore represent a highly quantitative assay for on-target NAMPT inhibition in EWS that could potentially be incorporated in future clinical trials.

Intermittently treated tumors retained sensitivity to OT-82, however, sustained durability of response was not observed using an intermittent schedule. This appears to be consistent with the data describing single-agent OT-82 use in most in vivo leukemia models, although the results of prolonged intermittent dosing were not reported in those studies34,58.

These findings suggest that a subpopulation of cells may be resistant, although the underlying mechanisms responsible for this are not yet clear. Tumor heterogeneity, incomplete or inconsistent drug delivery to certain areas of the tumor, or apoptotic resistance, all of which have been described as mechanisms of resistance for other anticancer agents65–67 may contribute to our understanding of these results and are important future areas of investigation.

The addition of DNA damage-augmenting agents (irinotecan and niraparib), which are clinically relevant agents for EWS, enhanced both disease control and animal survival in our models, suggesting that clinically, OT-82 would optimally be used as part of a combination regimen.

While single-agent OT-82 and OT-82 plus niraparib both resulted in tumor regression in the in vivo models tested, the addition of low-dose irinotecan to OT-82 slowed tumor growth but surprisingly did not result in tumor regressions. This may suggest that a higher dose of irinotecan is required for such an effect and/or that the drug dosing schedule and/or sequencing of agents may be important.

In addition, while our experiments used clinically relevant doses of irinotecan and niraparib that were guided by existing human pharmacokinetic (PK) data, human PK data on OT-82 are not yet available. These forthcoming data represent an additional important factor in assessing the translational potential of OT-82.

Further preclinical investigation should focus on establishing and optimizing effective combination regimens using clinically achievable drug concentrations that result in durable responses in preclinical models. Consideration of the human toxicity profile of OT-82 and whether it overlaps with the toxicity profiles of potential combination partners will also be of utmost importance.

In conclusion, our results demonstrate that dependence on NAMPT represents a therapeutic vulnerability in EWS, and thus, that OT-82, with its improved toxicity profile, may be a promising new agent for the treatment of EWS. These findings provide a rationale for early phase testing of this drug in this patient population.

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481307/


More information: Yi-Ru Yu et al, Disturbed mitochondrial dynamics in CD8+ TILs reinforce T cell exhaustion, Nature Immunology (2020). DOI: 10.1038/s41590-020-0793-3

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