K. Krukowski

The anti-diabetic drug metformin protects against chemotherapy-induced peripheral neuropathy in a mouse model.

Chemotherapy-induced peripheral neuropathy (CIPN) characterized by loss of sensory sensitivity and pain in hands and feet is the major dose-limiting toxicity of many chemotherapeutics. At present, there are no FDA-approved treatments for CIPN. The anti-diabetic drug metformin is the most widely used prescription drug in the world and improves glycemic control in diabetes patients. There is some evidence that metformin enhances the efficacy of cancer treatment. The aim of this study was to test the hypothesis that metformin protects against chemotherapy-induced neuropathic pain and sensory deficits. Mice were treated with cisplatin together with metformin or saline. Cisplatin induced increased sensitivity to mechanical stimulation (mechanical allodynia) as measured using the von Frey test. Co-administration of metformin almost completely prevented the cisplatin-induced mechanical allodynia. Co-administration of metformin also prevented paclitaxel-induced mechanical allodynia. The capacity of the mice to detect an adhesive patch on their hind paw was used as a novel indicator of chemotherapy-induced sensory deficits. Co-administration of metformin prevented the cisplatin-induced increase in latency to detect the adhesive patch indicating that metformin prevents sensory deficits as well. Moreover, metformin prevented the reduction in density of intra-epidermal nerve fibers (IENFs) in the paw that develops as a result of cisplatin treatment. We conclude that metformin protects against pain and loss of tactile function in a mouse model of CIPN. The finding that metformin reduces loss of peripheral nerve endings indicates that mechanism underlying the beneficial effects of metformin includes a neuroprotective activity. Because metformin is widely used for treatment of type II diabetes, has a broad safety profile, and is currently being tested as an adjuvant drug in cancer treatment, clinical translation of these findings could be rapidly achieved.

Mechanisms of chemotherapy-induced behavioral toxicities

While chemotherapeutic agents have yielded relative success in the treatment of cancer, patients are often plagued with unwanted and even debilitating side-effects from the treatment which can lead to dose reduction or even cessation of treatment. Common side effects (symptoms) of chemotherapy include (i) cognitive deficiencies such as problems with attention, memory and executive functioning; (ii) fatigue and motivational deficit; and (iii) neuropathy. These symptoms often develop during treatment but can remain even after cessation of chemotherapy, severely impacting long-term quality of life. Little is known about the underlying mechanisms responsible for the development of these behavioral toxicities, however, neuroinflammation is widely considered to be one of the major mechanisms responsible for chemotherapy-induced symptoms. Here, we critically assess what is known in regards to the role of neuroinflammation in chemotherapy-induced symptoms. We also argue that, based on the available evidence, neuroinflammation is unlikely the only mechanism involved in the pathogenesis of chemotherapy-induced behavioral toxicities. We evaluate two other putative candidate mechanisms. To this end we discuss the mediating role of damage-associated molecular patterns (DAMPs) activated in response to chemotherapy-induced cellular damage. We also review the literature with respect to possible alternative mechanisms such as a chemotherapy-induced change in the bioenergetic status of the tissue involving changes in mitochondrial function in relation to chemotherapy-induced behavioral toxicities. Understanding the mechanisms that underlie the emergence of fatigue, neuropathy, and cognitive difficulties is vital to better treatment and long-term survival of cancer patients.

CD8+ T cells and endogenous IL-10 are required for resolution of chemotherapy-induced neuropathic pain

Chemotherapy-induced peripheral neuropathy (CIPN), characterized by pain and numbness in hands and feet, is a common side effect of cancer treatment. In most patients, symptoms of CIPN subside after treatment completion. However, in a substantial subgroup, CIPN persists long into survivorship. Impairment in pain resolution pathways may explain persistent CIPN. We investigated the contribution of T cells and endogenous interleukin (IL)-10 to resolution of CIPN. Paclitaxel-induced mechanical allodynia was prolonged in T-cell-deficient (Rag1−/−) mice compared with wild-type (WT) mice. There were no differences between WT and Rag1−/− mice in severity of paclitaxel-induced mechanical allodynia. Adoptive transfer of either CD3+ or CD8+, but not CD4+, T cells to Rag1−/− mice normalized resolution of CIPN. Paclitaxel treatment increased the number of T cells in lumbar dorsal root ganglia (DRG), where CD8+ T cells were the major subset. Inhibition of endogenous IL-10 signaling by intrathecal injection of anti-IL-10 to WT mice or Rag1−/− mice reconstituted with CD8+ T cells delayed recovery from paclitaxel-induced mechanical allodynia. Recovery was also delayed in IL-10 knock-out mice. Conversely, administration of exogenous IL-10 attenuated paclitaxel-induced allodynia. In vitro, IL-10 suppressed abnormal paclitaxel-induced spontaneous discharges in DRG neurons. Paclitaxel increased DRG IL-10 receptor expression and this effect requires CD8+ T cells. In conclusion, we identified a novel mechanism for resolution of CIPN that requires CD8+ T cells and endogenous IL-10. We propose that CD8+ T cells increase DRG IL-10 receptor expression and that IL-10 suppresses the abnormal paclitaxel-induced spontaneous discharges by DRG neurons to promote recovery from CIPN. SIGNIFICANCE STATEMENT Chemotherapy-induced peripheral neuropathy persists after completion of cancer treatment in a significant subset of patients, whereas others recover. Persistent neuropathy after completion of cancer treatment severely affects quality of life. We propose that understanding how neuropathy resolves will identify novel avenues for treatment. We identified a novel and critical role for CD8+ T cells and for endogenous IL-10 in recovery from paclitaxel-induced neuropathy in mice. Enhancing the capacity of CD8+ T cells to promote resolution or increasing IL-10 signaling are promising targets for novel interventions. Clinically, peripheral blood CD8+ T-cell function and/or the capacity of individuals to produce IL-10 may represent biomarkers of risk for developing persistent peripheral neuropathy after completion of cancer treatment.

Prevention of chemotherapy-induced peripheral neuropathy by the small-molecule inhibitor pifithrin-μ.

Abstract Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of cancer treatment. It is the most frequent cause of dose reduction or treatment discontinuation in patients treated for cancer with commonly used drugs including taxanes and platinum-based compounds. No FDA-approved treatments for CIPN are available. In rodents, CIPN is represented by peripheral mechanical allodynia in association with retraction of intraepidermal nerve fibers. The mechanism of chemotherapy-induced neurotoxicity is unclear, but it has been established that mitochondrial dysfunction is an important component of the dysregulation in peripheral sensory neurons. We have shown earlier that inhibition of mitochondrial p53 accumulation with the small compound pifithrin-&mgr; (PFT-&mgr;) prevents cerebral neuronal death in a rodent model of hypoxic-ischemic brain damage. We now explore whether PFT-&mgr; is capable of preventing neuronal mitochondrial damage and CIPN in mice. We demonstrate for the first time that PFT-&mgr; prevents both paclitaxel- and cisplatin-induced mechanical allodynia. Electron microscopic analysis of peripheral sensory nerves revealed that PFT-&mgr; secured mitochondrial integrity in paclitaxel-treated mice. In addition, PFT-&mgr; administration protects against chemotherapy-induced loss of intraepidermal nerve fibers in the paw. To determine whether neuroprotective treatment with PFT-&mgr; would interfere with the antitumor effects of chemotherapy, ovarian tumor cells were cultured in vitro with PFT-&mgr; and paclitaxel. Pifithrin-&mgr; does not inhibit tumor cell death but even enhances paclitaxel-induced tumor cell death. These data are the first to identify PFT-&mgr; as a potential therapeutic strategy for prevention of CIPN to combat one of the most devastating side effects of chemotherapy.

Inhibition of Mitochondrial p53 Accumulation by PFT-μ Prevents Cisplatin-Induced Peripheral Neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN), a debilitating major side effect of cancer treatment, is characterized by pain and sensory loss in hand and feet. Platinum-based chemotherapeutics like cisplatin frequently induce CIPN. The molecular mechanism underlying these neurotoxic symptoms is incompletely understood and there are no preventive or curative interventions. We hypothesized that cisplatin acts as a cellular stressor that triggers p53 accumulation at mitochondria, leading to mitochondrial dysfunction and CIPN. To test this hypothesis, we examined the effect of the small molecule pifithrin-μ (PFT-μ), an inhibitor of p53 mitochondrial association on CIPN and the associated mitochondrial dysfunction. We show here for the first time that in vivo cisplatin rapidly increases mitochondrial accumulation of p53 in dorsal root ganglia (DRG), spinal cord, and peripheral nerve without evidence for apoptosis. Cisplatin-treatment also reduced mitochondrial membrane potential and lead to abnormal mitochondrial morphology and impaired mitochondrial function in DRG neurons. Pre-treatment with PFT-μ prevented the early cisplatin-induced increase in mitochondrial p53 and the reduction in mitochondrial membrane potential. Inhibition of the early mitochondrial p53 accumulation by PFT-μ also prevented the abnormalities in mitochondrial morphology and mitochondrial bioenergetics (reduced oxygen consumption rate, maximum respiratory capacity, and adenosine triphosphate synthesis) that develop in DRG and peripheral nerve after cisplatin-treatment. Functionally, inhibition of mitochondrial p53 accumulation prevented the hallmarks of CIPN including mechanical allodynia, peripheral sensory loss (numbness) as quantified by an adhesive-removal task, and loss of intra-epidermal nerve fibers. In conclusion, PFT-μ is a potential neuroprotective agent that prevents cisplatin-induced mitochondrial dysfunction in DRG and peripheral nerves thereby protecting against CIPN through blockade of the early cisplatin-induced increase in mitochondrial p53. Notably, there is accumulating evidence that PFT-μ has anti-tumor activities and could therefore be an attractive candidate to prevent CIPN while promoting tumor cell death.

HDAC6 inhibition effectively reverses chemotherapy-induced peripheral neuropathy

Chemotherapy-induced peripheral neuropathy is one of the most common dose-limiting side-effects of cancer treatment. Currently, there is no FDA-approved treatment available. Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase whose function includes regulation of α -tubulin- dependent intracellular mitochondrial transport. Here we examined the effect of HDAC6 inhibition on cisplatin-induced peripheral neuropathy. We used a novel HDAC6 inhibitor ACY-1083, which shows 260-fold selectivity towards HDAC6 versus other HDACs. Our results show that HDAC6 inhibition completely reversed already existing cisplatin-induced mechanical allodynia, spontaneous pain, and numbness. These findings were confirmed using the established HDAC6 inhibitor ACY-1215 (Ricolinostat), which is currently in clinical trials for cancer treatment. Mechanistically, treatment with the HDAC6 inhibitor increased α -tubulin acetylation in the peripheral nerve. In addition, HDAC6 inhibition restored the cisplatin-induced reduction in mitochondrial bioenergetics and mitochondrial content in the tibial nerve, indicating increased mitochondrial transport. At a later time point, dorsal root ganglion mitochondrial bioenergetics also improved. HDAC6 inhibition restored the loss of intra-epidermal nerve fiber density in cisplatin-treated mice. Our results demonstrate that pharmacological inhibition of HDAC6 completely reverses all the hallmarks of established cisplatin-induced peripheral neuropathy. These results are especially promising because one of the HDAC6 inhibitors tested here is currently in clinical trials as an add-on cancer therapy, highlighting the potential for a fast clinical translation of our findings.

Abstract # 1720 T lymphocytes are required for resolution of inflammatory pain and depression-like behavior

Transient pain and depressed mood commonly develop in response to inflammation. While normally reversible once the peripheral inflammation has resolved, depression and pain persist in some individuals. On the basis of previous studies showing that T lymphocytes contribute to homeostasis and repair in the nervous system, we hypothesized that T lymphocytes promote endogenous resolution of comorbid depression-like behavior and pain. Transient pain and depression-like behavior were induced by systemic administration of lipopolysaccharide (LPS) or by intraplantar injection of complete Freund’s adjuvant (CFA). In both models, depression-like behavior, mechanical allodynia and ongoing pain (measured by analgesic-induced conditioned place preference) were substantially prolonged in mice lacking mature lymphocytes (Rag1 and Rag2 KO mice). Reconstitution of Rag KO mice with T cells normalized resolution of depression-like behavior, mechanical allodynia and ongoing pain. Increased expression of the tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO1) in the brain which mediates depression-like behavior in response to peripheral inflammation was also prolonged in absence of T cells. The anti-inflammatory cytokine IL-10 and the ATP-degrading enzyme CD73 were upregulated in the prefrontal cortex (PFC) of WT mice after they had recovered, but not in Rag KO mice at the same time point. These findings demonstrate an important role for T lymphocytes in the endogenous resolution of inflammatory pain and depression.

An HDAC6 inhibitor for treatment of chemotherapy-induced peripheral numbness and pain in a mouse model

Neurotoxic side-effects of chemotherapy, including pain and numbness in hands and feet, frequently leads to dose reduction. This chemotherapy-induced peripheral neuropathy (CIPN) often persists long into survivorship and negatively affects quality of life. No drugs are available to prevent or treat CIPN and underlying mechanisms are only partially understood. Animal models for investigating chemotherapy-induced numbness are lacking. We have developed a tool for measuring numbness in mice; the adhesive removal task (ART). This task measures the time it takes an animal to show a behavioral response to an adhesive patch on its paw. We show that cisplatin induces mechanical hyperalgesia and impairs performance in the ART without altering general locomotor activity or motor coordination. Here we investigated the capacity of a specific histone deacetylase 6 (HDAC6) inhibitor to treat cisplatin-induced numbness and pain in mice. We show that the HDAC6 inhibitor, ACY-1083, effectively treats cisplatin-induced numbness. ACY-1083 also reverses existing cisplatin-induced mechanical hyperalgesia. ACY-1083 alone did not affect sensitivity to mechanical stimulation or performance in the ART. Analysis of cultured dorsal root ganglia revealed that cisplatin treatment decreased mitochondrial axonal transport, while co-culture with ACY-1083 attenuated this decrease. This is important because HDAC6 acts by deacetylating alpha-tubulin, which is thought to promote axonal transport. To our knowledge this is the first identified drug capable of treating already existing chemotherapy-induced numbness and pain.

Prevention of chemotherapy-induced peripheral neuropathy by the small-molecule inhibitor Pifithrin-mu in a mouse model

Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of cancer treatment and a frequent cause of treatment discontinuation. No FDA-approved treatments for CIPN are available. In rodents CIPN is represented by peripheral mechanical hyperalgesia in association with retraction of intra-epidermal nerve fibers (IENFs). The mechanism of chemotherapy-induced neurotoxicity is unclear but it has been established that mitochondrial dysfunction is an important component of the dysregulation. We have shown earlier that inhibition of mitochondrial p53 accumulation with the small compound pifithrin-mu (PFT-mu) prevents cerebral neuronal death in a rodent model of hypoxic-ischemic brain damage. We now explored whether PFT- mu is capable of preventing neuronal mitochondrial damage and CIPN in mice. We demonstrate for the first time that PFT-mu prevents both paclitaxel- and cisplatin-induced mechanical hyperalgesia. Electron microscopic analysis of peripheral sensory nerves revealed that PFT-mu secured mitochondrial integrity in paclitaxel-treated mice. In addition, PFT-mu administration protected against chemotherapy-induced loss of IENFs in the paw. To determine whether neuroprotective treatment with PFT-mu would interfere with the anti-tumor effects of chemotherapy, ovarian tumor cells were cultured in vitro with PFT-mu and paclitaxel. The results showed that PFT-mu does not inhibit tumor cell death but even enhances paclitaxel-induced tumor cell death. These data are the first to identify PFT-mu as a potential therapeutic strategy for prevention of CIPN to combat the devastating side effects of chemotherapy.

Identification of CD8+ T lymphocytes as a critical step in the recovery from paclitaxel-induced peripheral neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) characterized by pain and numbness in hands and feet is a common side-effect of cancer treatment. In most patients symptoms of CIPN subside after treatment completion; however, in a substantial group CIPN can persist into survivorship. Mechanism(s) that regulate recovery from CIPN have not been studied. We investigated whether T lymphocytes play a role in regulating CIPN recovery. In WT and Rag1−/− mice mechanical hyperalgesia developed immediately following paclitaxel treatment but was significantly prolonged in Rag1−/− mice. When Rag1−/− mice were reconstituted with CD3+ T cells recovery was measured at rates comparable to WT mice. Next, we investigated if T lymphocytes are present in lumbar dorsal root ganglia (DRGs) that innervate the hind paws where hyperalgesia is measured. Increased numbers of CD3+ T lymphocytes were detected in lumbar DRGs of paclitaxel-treated mice when compared to vehicle-treated mice on day 7 and the majority were CD8+ T lymphocytes. Reconstitution of Rag1−/− mice with either CD8+ or CD4+ lymphocytes showed that only those mice that received CD8+ lymphocytes recovered from paclitaxel-induced neuropathy. Furthermore, blockade of IL-10 by intrathecal injection of anti-IL-10 antibody delayed recovery. Down-regulation of peripheral monocyte recruitment by anti-CCR2 antibody inhibited CD8+ T lymphocyte-mediated recovery as well suggesting that the interplay between CD8+ lymphocytes and monocytes is crucial for recovery from pac litaxel-induced neuropathy.