Differential Morphological and Biochemical Recovery from Chemotherapy-Induced Peripheral Neuropathy Following Paclitaxel, Ixabepilone, or Eribulin Treatment in Mouse Sciatic Nerves.

The reversibility of chemotherapy-induced peripheral neuropathy (CIPN), a disabling and potentially permanent side effect of microtubule-targeting agents (MTAs), is becoming an increasingly important issue as treatment outcomes improve. The molecular mechanisms regulating the variability in time to onset, severity, and time to recovery from CIPN between the common MTAs paclitaxel and eribulin are unknown. Previously (Benbow et al. in Neurotox Res 29:299-313, 2016), we found that after 2 weeks of a maximum tolerated dose (MTD) in mice, paclitaxel treatment resulted in severe reductions in axon area density, higher frequency of myelin abnormalities, and increased numbers of Schwann cell nuclei in sciatic nerves. Biochemically, eribulin induced greater microtubule-stabilizing effects than paclitaxel. Here, we extended these comparative MTD studies to assess the recovery from these short-term effects of paclitaxel, eribulin, and a third MTA, ixabepilone, over the course of 6 months. Paclitaxel induced a persistent reduction in axon area density over the entire 6-month recovery period, unlike ixabepilone- or eribulin-treated animals. The abundance of myelin abnormalities rapidly declined after cessation of all drugs but recovered most slowly after paclitaxel treatment. Paclitaxel- and ixabepilone- but not eribulin-treated animals exhibited increased Schwann cell numbers during the recovery period. Tubulin composition and biochemistry rapidly returned from MTD-induced levels of α-tubulin, acetylated α-tubulin, and end-binding protein 1 to control levels following cessation of drug treatment. Taken together, sciatic nerve axons recovered more rapidly from morphological effects in eribulin- and ixabepilone-treated animals than in paclitaxel-treated animals and drug-induced increases in protein expression levels following paclitaxel and eribulin treatment were relatively transient.

Targeting the PI3K/Akt pathway in murine MDS/MPN driven by hyperactive Ras.

Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myelodysplastic/myeloproliferative neoplasia (MDS/MPN) overlap syndromes that respond poorly to conventional treatments. Aberrant Ras activation because of NRAS, KRAS, PTPN11, CBL and NF1 mutations is common in CMML and JMML. However, no mechanism-based treatments currently exist for cancers with any of these mutations. An alternative therapeutic strategy involves targeting Ras-regulated effector pathways that are aberrantly activated in CMML and JMML, which include the Raf/MEK/ERK and phosphoinositide-3'-OH kinase (PI3K)/Akt cascades. Mx1-Cre, Kras(D12) and Mx1-Cre, Nf1(flox/)(-) mice accurately model many aspects of CMML and JMML. Treating Mx1-Cre, Kras(D12) mice with GDC-0941 (also referred to as pictilisib), an orally bioavailable inhibitor of class I PI3K isoforms, reduced leukocytosis, anemia and splenomegaly while extending survival. However, GDC-0941 treatment attenuated activation of both PI3K/Akt and Raf/MEK/ERK pathways in primary hematopoietic cells, suggesting it could be acting through suppression of Raf/MEK/ERK signals. To interrogate the importance of the PI3K/Akt pathway specifically, we treated mice with the allosteric Akt inhibitor MK-2206. This compound had no effect on Raf/MEK/ERK signaling, yet it also induced robust hematologic responses in Kras and Nf1 mice with MPN. These data support investigating PI3K/Akt pathway inhibitors as a therapeutic strategy in JMML and CMML patients.

Prostate-specific membrane antigen (PSMA) enzyme activity is elevated in prostate cancer cells.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a glutamate carboxypeptidase that cleaves terminal carboxy glutamates from both the neuronal dipeptide N-acetylaspartylglutamate (NAAG) and gamma-linked folate polyglutamate. The prostate enzyme has activity in both the membrane and cytosolic fractions termed PSMA and PSMA', respectively. METHODS: Using a NAAG hydrolytic radioenzymatic assay, we quantitated the enzymatic activity of PSMA and PSMA' in normal, benign prostatic hyperplasia (BPH), and prostate cancer (PC) tissues from radical prostatectomies. PSMA enzyme activity was evaluated in each tissue type and expressed per milligram protein and epithelial cell content. RESULTS: PSMA and PSMA' enzyme activities were significantly elevated in prostate cancer when compared to normal prostate tissue and BPH. Ratios of PSMA to PSMA' were also decreased in BPH as compared to cancerous and normal tissue. CONCLUSIONS: Prostate carcinogenesis is associated with an elevation in PSMA and PSMA' enzyme activity. In contrast, no such enhancement in PSMA activity is observed with benign neoplastic changes in BPH. Thus, the enhancement observed in prostate cancer is not simply related to a generalized prostatic hyperplasia, but is specific to its malignancy.

Suramin potently inhibits the enzymatic activity of PSM.

BACKGROUND: The polysulfonated napthlyurea suramin has shown significant antitumor activity in patients with hormone-refractory metastatic prostate cancer. The mechanism by which suramin exerts this effect is unknown. In 1993, prostate-specific membrane antigen (PSM) was identified as a prostate biomarker that is elevated in hormone-refractory and metastatic prostate cancer. PSM is a glutamate exocarboxypeptidase capable of cleaving the terminal alpha-linked glutamate from the dipeptide N-acetyl-aspartyl-glutamate (NAAG) and the gamma-linked glutamates from folate polyglutamate. METHODS: Using a NAAG hydrolytic radioenzymatic assay, we tested whether suramin had any effect on the enzymatic activity of PSM. RESULTS: We demonstrate that suramin potently inhibits the enzymatic activity of PSM with a K(i) = 15 nM and 68 nM for the membrane-associated and soluble forms of PSM, respectively. In addition, we show that suramin inhibition of PSM enzyme activity displays the kinetics of a classic competitive inhibitor. CONCLUSIONS: This is one of the most potent activities described for suramin to date and may represent a portion of its pharmacologic and/or toxicological mechanism of action.

Characterization of the enzymatic activity of PSM: comparison with brain NAALADase.

BACKGROUND: The prostate cancer marker prostate-specific membrane antigen (PSM) is highly homologous to the brain enzyme N-acetylated alpha-linked acidic dipeptidase (NAALADase). NAALADase is known to cleave terminal carboxy glutamates from both the neuronal peptide N-acetylaspartylglutamate (NAAG) and folate polyglutamate. In this report, we compare the NAAG hydrolyzing activity of NAALADase and the prostate enzyme PSM. METHODS: Using a NAAG hydrolytic radioenzymatic assay, we compared the pharmacological and kinetic properties of the brain and prostate enzymes. RESULTS: Eight normal prostate tissues from different species exhibited NAAG hydrolyzing activity. Among 14 cancer cell lines examined, activity was observed in human LNCaP, PC-82, and rat Dunning G and AT-1 cells. Brain exhibited membrane-localized activity exclusively, while the prostate enzyme had activity in both membrane and cytosolic fractions. The only observed pharmacological difference was the sensitivity to their putative substrates, folate polyglutamate and NAAG. Kinetically, the soluble form of the prostate enzyme had two catalytic sites, while the membrane-bound form exhibited single site kinetics with a lower Vmax than the brain enzyme, which may suggest a less active hydrolase in the prostate. CONCLUSIONS: The brain enzyme NAALADase and the prostate enzyme PSM are remarkably similar. The importance of the differences in substrate specificities and kinetic parameters remains to be elucidated.