AMPK activator metformin shown for the first time to destabilize latent HIV-1 reservoir in chronic HIV-1 patients & lower immune checkpoint PD-1

Goldsmith Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus [Public domain], via Wikimedia Commons; CC BY 2.5 (http://creativecommons.org/licenses/by/2.5)], via Wikimedia Commons; By NASA [Public domain], via Wikimedia

At the International AIDS Society’s (IAS) HIV Cure and Cancer Forum held in Paris, France in July of 2017, researchers from the University of Hawaii demonstrated for the first time that the anti-diabetic drug and AMPK activator metformin not only decreased the percentage of CD4+ T cells expressing the immune checkpoint receptors PD-1, TIGIT, and TIM-3 but also destabilized the viral reservoir in chronically-infected HIV-1 patients, indicating that metformin may indeed contribute to HIV-1 eradication by reactivating CD4+ T cells latently-infected with HIV-1 [1-3]. Current antiretroviral drugs selectively targets only replicating viruses capable of inducing viral gene expression in activated CD4+ T cells [4]. However, a method known as the “shock and kill” approach in HIV-1 cure research involves the reactivation of a small subset of primarily CD4+ memory T cells latently infected with HIV-1 (dormant) with various agents/drugs (i.e. “shock”) and is thought to facilitate virus-induced cell death or immune recognition and destruction of the virus (i.e. “kill”) [4]. Immune checkpoint receptors including PD-1 are considered markers of T cell exhaustion and have also previously been found to be positively associated with T cells that harbor latent HIV-1 [5,6].

As the inhibition of PD-1/PD-L1 significantly enhances CD8+ T cell-mediated immunological responses to both viruses and cancer cells, the finding that metformin decreases PD-1 and destabilizes the latent viral reservoir in chronically-infected HIV-1 patients provides additional evidence and supports several recent publications in which I proposed for the first time that AMPK, which is critical for T cell activation, links the reactivation of latent HIV-1 with the differentiation and/or apoptosis of cancer stem cells and the amelioration of accelerated cellular aging defects in Hutchinson-Gilford progeria syndrome (HGPS) [4,7-10]. Because metformin has recently been shown to improve accelerated aging defects in cells derived from HGPS patients, decrease PD-1 expression on T cells, and potently induce cancer stem cell differentiation and/or apoptosis (e.g. glioblastoma), AMPK activation may indeed lead to the inhibition of tumorigenesis, viral eradication, and mitigation of accelerated aging [1,11-13].
    
At the IAS HIV Cure and Cancer Forum, Chew et al. presented data from an open label, 24 week clinical trial in which metformin (500 mg increasing to 1000 mg at week 4) was administered to 12 HIV-1 positive patients who were on antiretroviral therapy for greater than a year with plasma HIV RNA less than 50 copies/ml [1]. A non-metformin treated observational arm was also included. Compared to the observational arm, metformin-treated patients exhibited a significant 24 week decease in the change of single positive PD-1+, dual expressing TIGIT+PD-1+, and triple expressing TIGIT+PD-1+TIM-3+ CD4 T cells [1]. Strikingly, although integrated HIV DNA (an indicator of HIV-1 latency) in T cells remained remarkably stable in the 3 subjects in the observation arm, a variance in integrated HIV DNA was observed in metformin-treated patients, indicating that the viral reservoir had been destabilized. Interestingly, the authors also noted a statistically significant decrease in CD32a in metformin-treated patients [1]. CD32a is a surface receptor protein in immune cells that has recently been demonstrated as a reliable marker of CD4+ T cell reservoirs in HIV-1 infected patients harboring replication-competent proviruses, providing compelling evidence that metformin-induced AMPK activation may promote viral eradication by inducing reactivation of latent HIV-1, as evidenced by reductions in PD-1, TIGIT, TIM-3, and CD32a, all markers of CD4+ T cell latent HIV-1 reservoirs [1,14].

In addition to inducing latent HIV-1 reactivation, metformin-induced AMPK activation may also mitigate T cell exhaustion and enhance the immunological response of cytotoxic CD8+ T cells to viruses and cancer cells. Indeed, PD-L1, a ligand that binds to the PD-1 receptor on T cells, has been found on breast and colon cancer stem cells and metformin, AICAR (an AMPK activator), and rapamycin (a macrolide that activates AMPK in vivo) have each been shown to decrease PD-L1 expression on human lung cancer cells [15-18]. Intriguingly, metformin has also been shown to improve intratumoral T cell function and tumor clearance in mice by potentiating PD-1 blockade and significantly increasing the number of activated CD8+ T cells [19].

Moreover, as AMPK is critical for T cell activation and AMPK inhibition during T cell activation leads to T cell death, recent studies indicating that AMPK activators decrease PD-1 expression on T cells and enhance CD8+ T cell-mediated immunological responses strongly suggests that AMPK represents a central node linking latent HIV-1 reactivation with T cell-mediated virus and cancer cell elimination (i.e. immunotherapy) [20]. Eikawa et al. for example showed that metformin enabled normal mice (but not T cell-deficient mice) to reject solid tumors by increasing the number of CD8+ tumor-infiltrating lymphocytes (TILs) and protecting them from exhaustion in an AMPK-dependent manner [21]. Perhaps most convincingly, Chamoto et al. demonstrated in a model using mouse colon cancer MC38 cells that PD-1 blockade (using a PD-L1 inhibitor) led to activation of mitochondria in tumor-reactive cytotoxic CD8+ T lymphocytes, evidenced by increased mitochondrial ROS (i.e. superoxide), larger mitochondrial mass, and higher mitochondrial membrane potential [22].

As mitochondria-derived ROS have previously been shown to be critical, if not indispensable for T cell activation, the combination of a PD-L1 inhibitor/mAb with Luperox (a ROS precursor) greatly enhanced the antitumor activity and survival of tumor-bearing mice [22,23]. Interestingly, the mitochondrial uncouplers FCCP and DNP also extended the survival time of PD-L1 mAb-treated animals, which was mitigated by the ROS scavenger MnTBAP [22]. As ROS have been independently shown to induce AMPK activation and AMPK is essential for T cell activation, the PD-L1 mAb alone induced AMPK activation and the combination of mitochondrial uncouplers with the PD-L1 mAb also further enhanced AMPK activation in CD8+ T cells, indicating that AMPK is essential for enhanced CD8+ T cell activation by PD-1 blockade, ROS generators, and mitochondrial uncouplers [4,22]. Indeed, the authors also showed that the AMPK activator A769662 further enhanced the antitumor activity by PD-L1 mAb treatment and improved animal survival. Additionally, oltipraz and bezafibrate, two compounds that also activate AMPK, strongly enhanced tumor-growth suppression and animal-survival by anti–PD-L1 treatment [22,24,25]. 

Such data, combined with the recent findings from the IAS HIV Cure and Cancer forum showing that metformin decreases the percentage of CD4+ T cells expressing PD-1, TIGIT, and TIM-3 and destabilizes the viral reservoir in chronically-infected HIV-1 patients, indicates that AMPK activation likely links the reactivation of latent HIV-1 with the facilitation of CD8+ T cell-mediated virus and cancer cell killing, potentially leading to HIV-1 eradication and cancer stem cell elimination, a hypothesis I initially proposed in May of 2017 [4].

Furthermore, AMPK activation also promotes oocyte meiotic induction and maturation (processes that are critical for efficient oocyte activation) and AMPK has recently been found localized across the entire acrosome in human spermatozoa [10,26,27]. The induction of cellular stress (e.g. increases in ROS, intracellular Ca2+, and/or AMP(ADP)/ATP ratio increase), which activates AMPK, also promotes oocyte meiotic induction/maturation, oocyte activation, and the acrosome reaction in human sperm, processes critical for the creation of all human life [26,28,29]. Indeed, the calcium ionophore ionomycin, which activates AMPK, is commonly used to promote latent HIV-1 reactivation and is extensively used to activate human oocytes, creating normal healthy children [29-31]. Such evidence further substantiates the novel and provocative assertion that AMPK activation links the amelioration of pathological cellular defects in Hutchinson-Gilford progeria syndrome with HIV-1 latency, adult and cancer stem cells, learning and memory, and the creation of all human life [4,8-10,32].

[Additional references for Figure above]: The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T. PLoS Biology Vol. 3/11/2005, e395; By NASA [Public domain], via Wikimedia Commons.

https://www.linkedin.com/pulse/ampk-activator-metformin-shown-first-time-destabilize-finley

References

1. G.M. Chew, D.C. Chow, S.A. Souza, et al. Impact of adjunctive metformin therapy on T cell exhaustion and viral persistence in a clinical trial of HIV-infected adults on suppressive ART. Journal of Virus Eradication 2017; 3 (Supplement 1): 6–19.
2. http://viruseradication.com/supplement-details/Abstracts_of_the_IAS_HIV_Cure_and_Cancer_Forum_2017/
3. http://www.iasociety.org/HIV-Programmes/Towards-an-HIV-Cure/Events/HIV-Cure-Cancer-Forum
4. Finley J. Elimination of cancer stem cells and reactivation of latent HIV-1 via AMPK activation: Common mechanism of action linking inhibition of tumorigenesis and the potential eradication of HIV-1. Med Hypotheses. 2017 Jul;104:133-146.
5. Fromentin R, Bakeman W, Lawani MB, et al. CD4+ T Cells Expressing PD-1, TIGIT and LAG-3 Contribute to HIV Persistence during ART. PLoS Pathog. 2016 Jul 14;12(7):e1005761.
6. Chew GM, Fujita T, Webb GM, et al. TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection. PLoS Pathog. 2016 Jan 7;12(1):e1005349.
7. Sakthivel P, Gereke M, Bruder D. Therapeutic intervention in cancer and chronic viral infections: antibody mediated manipulation of PD-1/PD-L1 interaction. Rev Recent Clin Trials. 2012 Feb;7(1):10-23.
8. Finley J. Alteration of splice site selection in the LMNA gene and inhibition of progerin production via AMPK activation. Med Hypotheses. 2014 Nov;83(5):580-7.
9. Finley J. Reactivation of latently infected HIV-1 viral reservoirs and correction of aberrant alternative splicing in the LMNA gene via AMPK activation: Common mechanism of action linking HIV-1 latency and Hutchinson-Gilford progeria syndrome. Med Hypotheses. 2015 Sep;85(3):320-32.
10. Finley J. Oocyte activation and latent HIV-1 reactivation: AMPK as a common mechanism of action linking the beginnings of life and the potential eradication of HIV-1. Med Hypotheses. 2016 Aug;93:34-47.
11. Park SK, Shin OS. Metformin alleviates ageing cellular phenotypes in Hutchinson-Gilford progeria syndrome dermal fibroblasts. Exp Dermatol. 2017 Feb 13. doi: 10.1111/exd.13323. [Epub ahead of print].
12. Egesipe AL, Blondel S, Cicero AL, et al. Metformin decreases progerin expression and alleviates pathological defects of Hutchinson-Gilford progeria syndrome cells. NPJ Aging Mech Dis. 2016 Nov 10;2:16026.
13. Sato A, Sunayama J, Okada M, et al. Glioma-initiating cell elimination by metformin activation of FOXO3 via AMPK. Stem Cells Transl Med 2012;1(11):811–24.
14. Descours B, Petitjean G, López-Zaragoza JL, et al. CD32a is a marker of a CD4 T-cell HIV reservoir harbouring replication-competent proviruses. Nature. 2017 Mar 23;543(7646):564-567.
15. Wu Y, Chen M, Wu P, Chen C, Xu ZP, Gu W. Increased PD-L1 expression in breast and colon cancer stem cells. Clin Exp Pharmacol Physiol. 2017 May;44(5):602-604.
16. Kurimoto R, Iwasawa S, Ebata T, et al. Drug resistance originating from a TGF-β/FGF-2-driven epithelial-to-mesenchymal transition and its reversion in human lung adenocarcinoma cell lines harboring an EGFR mutation. Int J Oncol. 2016 May;48(5):1825-36.
17. Lastwika KJ, Wilson W 3rd, Li QK, et al. Control of PD-L1 Expression by Oncogenic Activation of the AKT-mTOR Pathway in Non-Small Cell Lung Cancer. Cancer Res. 2016 Jan 15;76(2):227-38.
18. Chiao YA, Kolwicz SC, Basisty N, et al. Rapamycin transiently induces mitochondrial remodeling to reprogram energy metabolism in old hearts. Aging (Albany NY). 2016 Feb;8(2):314-27.
19. Scharping NE, Menk AV, Whetstone RD, Zeng X, Delgoffe GM. Efficacy of PD-1 Blockade Is Potentiated by Metformin-Induced Reduction of Tumor Hypoxia. Cancer Immunol Res. 2017 Jan;5(1):9-16.
20. Rao E, Zhang Y, Zhu G, et al. Deficiency of AMPK in CD8+ T cells suppresses their anti-tumor function by inducing protein phosphatase-mediated cell death. Oncotarget 2015;6(10):7944–58.
21. Eikawa S, Nishida M, Mizukami S, Yamazaki C, Nakayama E, Udono H. Immune-mediated antitumor effect by type 2 diabetes drug, metformin. Proc Natl Acad Sci U S A. 2015 Feb 10;112(6):1809-14.
22. Chamoto K, Chowdhury PS, Kumar A, et al. Mitochondrial activation chemicals synergize with surface receptor PD-1 blockade for T cell-dependent antitumor activity. Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):E761-E770.
23. Sena LA, Li S, Jairaman A, et al. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling. Immunity. 2013 Feb 21;38(2):225-36.
24. Zhong X, Xiu LL, Wei GH, et al. Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation. Acta Pharmacol Sin. 2011 May;32(5):591-600.
25. Kim TH, Eom JS, Lee CG, Yang YM, Lee YS, Kim SG. An active metabolite of oltipraz (M2) increases mitochondrial fuel oxidation and inhibits lipogenesis in the liver by dually activating AMPK. Br J Pharmacol. 2013 Apr;168(7):1647-61.
26. LaRosa C, Downs SM. Stress stimulates AMP-activated protein kinase and meiotic resumption in mouse oocytes. Biol Reprod. 2006 Mar;74(3):585-92.
27. Calle-Guisado V, de Llera AH, Martin-Hidalgo D, et al. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility. Asian J Androl. 2016 Sep 27. doi: 10.4103/1008-682X.185848. [Epub ahead of print].
28. de Lamirande E, Tsai C, Harakat A, Gagnon C. Involvement of reactive oxygen species in human sperm arcosome reaction induced by A23187, lysophosphatidylcholine, and biological fluid ultrafiltrates. J Androl. 1998 Sep-Oct;19(5):585-94.
29. Deemeh MR, Tavalaee M, Nasr-Esfahani MH. Health of children born through artificial oocyte activation: a pilot study. Reprod Sci. 2015 Mar;22(3):322-8.
30. Tamás P, Hawley SA, Clarke RG, et al. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes. J Exp Med. 2006 Jul 10;203(7):1665-70.
31. Spina CA, Anderson J, Archin NM, et al. An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog 2013;9(12):e1003834.
32. Finley J. Facilitation of hippocampal long-term potentiation and reactivation of latent HIV-1 via AMPK activation: Common mechanism of action linking learning, memory, and the potential eradication of HIV-1. Med Hypotheses. Manuscript submitted.