Hein, N., Bywater, M. J., Stanley, K., Verbrugge, I., Cullinane, C., Baker, A., Zuber, H., Rappaport, A., Drygin, D., Huser, N., Bliesath, J., Ryckman, D. M., Rice, W. G., Lowe, S. W., Johnstone, R. W., Pearson, R. B., McArthur, G. A., Hannan, R. D. (November 2011) Inhibition of RNA Polymerase I Transcription by CX-5461 As a Therapeutic Strategy for the Cancer-Specific Activation of p53 in MLL-Rearranged Acute Myeloid Leukemias. Blood, 118 (21). p. 1548. ISSN 0006-4971
Abstract
Increased transcription of the ribosomal genes (rDNA) by RNA Polymerase I (Pol I) is a common feature of human cancer1. Inhibition of Pol I transcription causes nucleolar stress that leads to the release of ribosomal proteins from the nucleolus into the nucleoplasm where they can sequester the p53 inhibitory protein MDM2, causing activation of p53 and induction of apoptosis2. We have developed a potent and selective small molecule inhibitor of Pol I transcription (CX-5461) that is non-genotoxic3. When evaluated for anti-proliferative activity against genetically diverse cancer cell lines, CX-5461 exhibited its greatest potency against wild-type (wt) p53 cells derived from hematological malignancies (median IC50 = 12 nM), while the median IC50s in cells derived from p53 mutated hematological, p53wt and p53 mutated solid tumors and normal cells were less sensitive to CX-5461 (median IC50s = 94 nM, 164 nM, 265 nM and 5 mM respectively), indicating CX-5461 selectively kills p53-wild type malignant hematopoietic cells. Consistent with the nucleolar stress model, p53 wt human leukemia cell lines exhibited robust activation of p53 signaling and apoptotic death in response to low nanomolar doses of CX-5461. To explore the therapeutic potential of Pol I transcription inhibition in vivo in hematological malignancies wt for p53 that are refractory to cytotoxic therapies we tested CX-5461 in mouse models of human acute myeloid leukemia (AML) expressing MLL fusion proteins4. Mice transplanted with 5×106 leukemia cells expressing MLL/ENL or MLL/AF9 together with oncogenic NRAS linked to GFP and luciferase biomarkers by virtue of bicistronic retroviral vectors, rapidly developed aggressive leukemia characterized by anemia, leukocytosis, hepatosplenomegaly and within 7–10 days (Mac1+, Gr-1) leukemic cell counts in peripheral blood. Treatment of recipient mice harboring MLL/ENL+NRAS leukemia with CX-5461 (40mg/kg Q3D) significantly increased median survival (17 days for vehicle vs 36 days for drug, P <0.0001), and reduced leukemic burden, as determined by leukemic cell luminescence imaging, peripheral white blood cell counts (20.77×103cells/ml±2.25 for vehicle vs 6.2×103cells/ml±0.68 for drug, P <0.0001) at sacrifice and FACS analysis of the peripheral blood (24.36% GFP leukemic cells±1.84 for vehicle vs 6.30% leukemic cells±0.5 for drug, P <0.0001) after 3 doses of treatment. The reduction in tumour burden was associated with in vivo activation of p53 signaling and apoptosis. In marked contrast, treatment of MLL/ENL+NRAS leukemic mice with a combination regimen of cytarabine (50 mg/kg 5XQD IP) and doxorubicin (1.5 mg/kg 3XQD IP) at the combined MTD dose failed to provide a significant survival advantage. Treatment of recipient mice harboring the highly aggressive MLL/AF9 + NRAS leukemia with CX-5461 (40mg/kg Q3D) was also able to increased overall survival (15 days for vehicle vs 23 days for drug, P 0.0009), with this delay accompanied by a significant decrease in leukemic burden as determined by peripheral white blood cell counts (114×103cells/ml±12.24 for vehicle vs 4.68×103cells/ml±0.7 for drug) and FACS analysis of the peripheral blood (65% GFP leukemic cells±4.9 for vehicle vs 0.26% leukemic cells±0.05 for drug) after 2 respectively 3 doses of treatment. Critically, in murine xenograft of human AML MV 4;11 that carries the MLL/AF4 fusion, treatment with CX-5461 (125 mg/kg Q7D IP) inhibited tumour growth by 93% and extended time to sacrifice from 21 to 36 days. Together this work demonstrates that CX-5461, which selectively inhibits Pol I transcription and non-genotoxically activates p53, may be used to therapeutically treat aggressive p53-wild type AMLs that are highly refractory to standard cytotoxic therapies. The fact that mutations or deletions of the p53 gene are relatively rare in AML, coupled with our data that p53-wt AML cancer cells are particularly sensitive to CX-5461, provides a compelling rationale for evaluating CX-5461 in this patient population.
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