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. 2011 Oct 13;118(15):4140-9.
doi: 10.1182/blood-2011-03-340323. Epub 2011 Aug 15.

HDM-2 inhibition suppresses expression of ribonucleotide reductase subunit M2, and synergistically enhances gemcitabine-induced cytotoxicity in mantle cell lymphoma

Richard J Jones  1 , Veerabhadran Baladandayuthapani, Sattva Neelapu, Luis E Fayad, Jorge E Romaguera, Michael Wang, Rakesh Sharma, Dajun Yang, Robert Z Orlowski
Affiliations

HDM-2 inhibition suppresses expression of ribonucleotide reductase subunit M2, and synergistically enhances gemcitabine-induced cytotoxicity in mantle cell lymphoma

Richard J Jones et al. Blood. .

Abstract

Mantle cell lymphoma (MCL) usually responds well to initial therapy but is prone to relapses with chemoresistant disease, indicating the need for novel therapeutic approaches. Inhibition of the p53 E3 ligase human homolog of the murine double minute protein-2 (HDM-2) with MI-63 has been validated as one such strategy in wild-type (wt) p53 models, and our genomic and proteomic analyses demonstrated that MI-63 suppressed the expression of the ribonucleotide reductase (RNR) subunit M2 (RRM2) VSports手机版. This effect occurred in association with induction of p21 and cell-cycle arrest at G(1)/S and prompted us to examine combinations with the RNR inhibitor 2',2'-difluoro-2'-deoxycytidine (gemcitabine). The regimen of MI-63-gemcitabine induced enhanced, synergistic antiproliferative, and proapoptotic effects in wtp53 MCL cell lines. Addition of exogenous dNTPs reversed this effect, whereas shRNA-mediated inhibition of RRM2 was sufficient to induce synergy with gemcitabine. Combination therapy of MCL murine xenografts with gemcitabine and MI-219, the in vivo analog of MI-63, resulted in enhanced antitumor activity. Finally, synergy was seen with MI-63-gemcitabine in primary patient samples that were found to express high levels of RRM2 compared with MCL cell lines. These findings provide a framework for translation of the rational combination of an HDM-2 and RNR inhibitor to the clinic for patients with relapsed wtp53 MCL. .

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Figures

Figure 1
Figure 1
HDM-2 inhibitors decrease RRM2 polypeptide expression. (A) REC-1, Granta-519, and JVM-2 wtp53 MCL cell lines were treated for 24 hours with either vehicle, 5μM MI-63, 5μM Nutlin, or 0.5μM doxorubicin (DOX) as a positive control for 24 hours. Qualitative PCR was performed to detect RRM2 and RRM2B mRNA levels, as well as β2M as a loading control, and transcripts were visualized by native gel electrophoresis. Lanes marked +RT received 1 μL of cDNA stock solution, whereas those labeled with 1:10 received 1 μL of 1:10 dilution of +RT. Representative images are shown in both panels of 1 of 3 independent experiments. (B) Protein levels of RRM2 and RRM2B, as well as β-actin as a loading control, were determined in lysates from MCL cells treated as described in panel A.
Figure 2
Figure 2
dFdC and MI-63 enhance cell death. (A) REC-1, Granta-519, and JVM-2, and the mutp53 MCL cell line JeKo-1, were treated for 24 hours with vehicle, 5μM MI-63, 10nM dFdC (GEM), or both. Flow cytometric analysis was then performed after staining with annexin-V and TO-PRO-3, from which the proportion of cells undergoing apoptosis was calculated and normalized to the vehicle control group. Values represent the mean ± SE from 3 independent experiments. An unpaired t test was performed to evaluate for significance; *P < .01 relative to MI-63 alone; #P < .01 relative to dFdC alone. (B) Granta-519 cells were incubated simultaneously with single agent MI-63 or dFdC for 72 hours. In parallel, cells were exposed either first to MI-63 for 24 hours followed by dFdC and MI-63 for 48 hours or to dFdC first for 24 hours followed later by MI-63 and dFdC for 48 hours. Cell viability was determined using the WST-1 reagent, and results are expressed as the percentage viability relative to the vehicle control, which was arbitrarily set at 100%. The presence of synergistic interactions was determined by calculation of the CI from the cell viabilities calculated across a serial dilution range of MI-63 or dFdC (Table 2). Each panel provides representative data from 1 of 3 independent experiments. (C) Protein levels of HDM-2, p53, RRM1, RRM2, RRM2B, and p21, and β-actin as a loading control, were determined by Western blotting of cellular lysates.
Figure 3
Figure 3
Excess dNTP reverses synergy between MI-63 and dFdC. (A) Granta-519 cells were incubated with vehicle, 5μM MI-63, 10nM dFdC (GEM), or both agents simultaneously for 24 hours, either without or with exogenous dNTPs at 50μM. Cell death was then determined by flow cytometry using annexin-V and TO-PRO-3 staining relative to the vehicle control. Each panel provides representative data from 1 of 3 independent experiments. An unpaired t test was performed comparing cells to which dNTPs had been added to those exposed to drug alone; *P < .05. (B) Cellular lysates were probed for PARP, HDM-2, p53, RRM2, RRM2B, and p21, as well as β-actin as a loading control. (C) REC-1 cells were infected with Lentiviral particles carrying a scrambled sequence shRNA or an shRNA targeting RRM2, and stable cell lines were generated by drug selection. Cellular lysates were then probed for their content of RRM2, RRM2B, and β-actin as a loading control. (D) REC-1 shRNA cells were incubated with vehicle, 5μM MI-63, 10nM dFdC, or both agents simultaneously for 24 hours, and the proportion of cells undergoing apoptosis was determined by flow cytometry using annexin-V and TO-PRO-3. Statistically significant differences are defined as *P < .05.
Figure 4
Figure 4
MI-63 and dFdC inhibit tumor growth in vivo. (A) Severe combined immunodeficiency mice were inoculated with Granta-519 cells subcutaneously and monitored until tumors were established. Five mice per group were then injected intraperitoneally with vehicle, MI-219 daily for 2 weeks, dFdC (GEM) every third day for 2 weeks, or both agents using the same schedules but at a 50% dose reduction. Tumor volumes were measured 3 times per week and are plotted as a function of time in the top panel. Statistically significant differences are defined as *P < .05 relative to the vehicle control and as #P < .05 relative to MI-219 alone. In the bottom panel, the average tumor growth rate per day was calculated, and the P values of each group are shown relative to the vehicle group, as well as to MI-219 alone, or dFdC alone.
Figure 5
Figure 5
Synergistic activity of MI-63 and dFdC in MCL patient samples. (A) MCL cells were purified from the peripheral blood of patients with circulating neoplastic cells using magnetic-activated cell sorting and CD19 microbeads. These cells were then either exposed to 5μM MI-63, 10nM dFdC, or both agents simultaneously for 24 hours. Cell death was determined by flow cytometry using annexin-V and TO-PRO-3 staining relative to the vehicle-treated control, and REC-1 cells were included as an additional control. Each panel provides representative data from 1 of 3 independent experiments, and *P < .05 denote significance relative to MI-63 alone, and #P < .05 denote significance relative to dFdC alone. (B) Aliquots of each of the primary samples analyzed in panel A also were subjected to RNA extraction, cDNA was synthesized, and the levels of RRM1, dCk, hENT-1, and RRM2 were measured by quantitative real-time PCR using the ΔΔCT method with the JVM-2 cell line used as a relative calibrator. The transcript level in REC-1 cells also was measured as a control and is plotted on a separate scale because of their high expression of RRM1, dCK, and hENT-1. (C) Real-time PCR analysis of RRM2 transcript levels in MCL patient samples is shown, along with REC-1 as a cell line control.
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V体育2025版 - References

    1. Campo E, Raffeld M, Jaffe ES. Mantle-cell lymphoma. Semin Hematol. 1999;36(2):115–127. - PubMed
    1. Martin P, Leonard JP. Novel therapeutic targets in mantle cell lymphoma. Expert Opin Ther Targets. 2007;11(7):929–940. - PubMed
    1. O'Connor OA. Mantle cell lymphoma: identifying novel molecular targets in growth and survival pathways. Hematology Am Soc Hematol Educ Program. 2007;2007:270–276. - PubMed
    1. Orlowski RZ, Stinchcombe TE, Mitchell BS, et al. Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies. J Clin Oncol. 2002;20(22):4420–4427. - PubMed
    1. O'Connor OA, Wright J, Moskowitz C, et al. Phase II clinical experience with the novel proteasome inhibitor bortezomib in patients with indolent non-Hodgkin's lymphoma and mantle cell lymphoma. J Clin Oncol. 2005;23(4):676–684. - PubMed

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