Mechanistic studies of APR-246 in leukemia

<p>PRIMA-1 and its analog APR-246 are novel drugs that restore the active conformation of mutated and unfolded p53 protein and induce apoptosis and cell death in various tumors in pre-clinical models. We first aimed to explore the effects of APR-246 alone and in combination with other drugs in acute myeloid leukemia (AML) in vitro. APR-246 induced dose-dependent apoptosis and increased active caspase-3 and p53 protein levels as well as the Bax/Bcl-2 ratio independently of TP53 mutational status. AML patient cells with TP53 mutations and complex karyotype were more resistant to conventional chemotherapeutic drugs but retained their sensitivity to APR-246. Pronounced synergism was found when combining APR-246 with DNR in AML patient cells and pre-incubation with APR-246 induced more synergistic effects compared to other treatment schedules in the AML cell line KMB3.</p><p>As APR-246 was shown to induce expression of genes protective of oxidative stress in global gene expression profiling, we furthermore aimed to study the effects of APR-246 on the redox status of AML cells. We confirmed that APR-246 increased ROS formation and depleted cells from glutathione. HO-1; a gene protecting from oxidative stress, was one of the most upregulated genes in response to APR-246. Both HO-1 and its transcriptional regulator NFE2L2 (Nrf2) were upregulated as detected by q-RT-PCR. APR-246 treatment induced Nrf2 activation by translocation of the Nrf2 protein from the cytosol to the nucleus. Transient knockdown of Nrf2 in KMB3 cells obliterated APR-246-induced up-regulation of HO-1 and increased its antitumoral effects. The PI3K inhibitor wortmannin and the mTOR inhibitor rapamycin; both up-stream regulators of Nrf2, inhibited APR-246-induced nuclear translocation of Nrf2 and induced synergism with APR-246.</p><p>A phase I first-in-man study including 22 patients with hematologic malignancies and prostate cancer was conducted. Dose escalations from 2 mg/kg to 90 mg/kg revealed a maximum tolerated dose (MTD) of 60 mg/kg and a half-life of 4-5 hours. The most common adverse effects were fatigue, dizziness, headache, and confusion. Dose limiting toxicities (DLTs) were increased ALT/AST (n=1), dizziness, confusion, and sensory disturbances (n=2). Tumor cells showed cell cycle arrest, increased apoptosis, and up-regulation of p53 target genes.</p><p>We finally showed that miR-34b/c is epigenetically silenced by DNA methylation in chronic lymphocytic leukemia (CLL). As being down-stream regulators of p53, miR-34b/c expression levels were induced by PRIMA-1 as well as by doxorubicin and decitabine. Over-expression of miR-34b/c in CLL cells increased apoptosis, which suggest a tumor suppressor function for these microRNAs.</p><p>In conclusion, AML cells are sensitive to APR-246 in vitro irrespectively of TP53 mutational status. The substance induces oxidative stress and activates the Nrf2/HO-1 protective pathway. In a first-in-man study, APR-246 was shown to be to have a favorable pharmacokinetic profile and to induce p53-dependent biologic effects in vivo. Either in combination with conventional chemotherapeutic drugs or PI3K inhibitors, synergistic antileukemic effects can be obtained which holds a promise for further combination studies in vivo.</p><h3>List of scientific papers</h3><p>I. Ali D, Jonsson-Videsäter K, Deneberg S, Bengtzen S, Nahi H, Paul C, Lehmann S. APR-246 exhibits anti-leukemic activity and synergism with conventional chemotherapeutic drugs in acute myeloid leukemia cells. Eur J Haematol. 2011 Mar; 86(3): 206-15. <br><a href="https://doi.org/10.1111/j.1600-0609.2010.01557.x">https://doi.org/10.1111/j.1600-0609.2010.01557.x</a><br><br> </p><p>II. Ali D, Mohammad D. K, Jonsson-Videsäter K, NoreB. F, PaulC, Lehmann S. APR-246-induced cytotoxicity is related to induction of oxidative stress and activation of the HO-1/NRF2 axis in leukemia cells. [Manuscript]</p><p>III. Lehmann S, Bykov VJ, Ali D, Andrén O, Cherif H, Tidefelt U, Uggla B, Yachnin J, Juliusson G, Moshfegh A, Paul C, Wiman KG, Andersson PO. Targeting p53 in vivo: a first-in-human study with p53-targeting compound APR-246 in refractory hematologic malignancies and prostate cancer. J Clin Oncol. 2012, Oct 10; 30(29): 3633-9. <br><a href="https://doi.org/10.1200/JCO.2011.40.7783">https://doi.org/10.1200/JCO.2011.40.7783</a><br><br> </p><p>IV. Deneberg S, Kanduri M, Ali D, Bengtzen S, Karimi M, Qu Y, Kimby E, Rosenquist R, Lennartsson A and Lehmann S. MicroRNA-34b/c is aberrantly hypermethylated by an epigenetic switch mechanism in B-cell Chronic Lymphatic Leukemia. [Manuscript]</p>