Letter to the Editor

Leukemia (2012) 26, 2421–2424; doi:10.1038/leu.2012.110; published online 15 May 2012

Systemic microRNA-34a delivery induces apoptosis and abrogates growth of diffuse large B-cell lymphoma in vivo

V J Craig1, A Tzankov2, M Flori1, C A Schmid1, A G Bader3 and A Müller1

  1. 1Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland
  2. 2Institute of Pathology, University of Basel, Basel, Switzerland
  3. 3Mirna Therapeutics, Inc., Austin, TX, USA

Correspondence: AG Bader, E-mail: abader@mirnarx.com (AGB); A Müller, E-mail: mueller@imcr.uzh.ch (AM)

Diffuse large B-cell lymphoma (DLBCL) accounts for ~30% of all B-cell lymphomas. The standard therapy consists of three chemotherapeutics (cyclophosphamide, doxorubicin and vincristine) combined with the steroid prednisone (CHOP) and/or the CD20-specific antibody rituximab.1 Two major subtypes of DLBCL are distinguished based on gene expression profiles that resemble those of activated B cells and germinal center B cells (ABC and GC types).2 We have reported recently that the expression of post-transcriptionally active small regulatory RNAs (microRNAs (miRNAs)) with tumor-suppressive properties3, 4, 5 is strongly deregulated in both nodal and extranodal DLBCL relative to indolent lymphomas arising at the same sites.6 All examined DLBCL cases exhibited a consistent signature of downregulated miRNAs independent of their location, which shared the common feature of being repressed by the transcription factor Myc. Myc was indeed found to be overexpressed in most analyzed cases of extranodal DLBCL, but not the corresponding indolent lymphomas. One miRNA in particular, miR-34a, emerged as a potent tumor-suppressor miRNA with the potential to inhibit the proliferation of various DLBCL cell lines.6 The tumor-suppressive activity of miR-34a could be attributed to the specific knockdown of FoxP1, a transcription factor and known target of miR-34a,6, 7 whose overexpression is associated with inferior prognosis in DLBCL patients.8

Here, we have examined the therapeutic benefit of miR-34a ‘replacement therapy’ in a xenograft model of DLBCL and have investigated the mechanistic basis underlying treatment efficacy. To this end, NOD/SCID/IL2Rγ/ mice were inoculated subcutaneously with U2932 cells, an ABC type DLBCL cell line with minimal endogenous miR-34a expression (Supplementary Figure 1a) owing to promoter hypermethylation (data not shown). Once palpable tumors of ~50mm3 had formed, the mice received intratumoral doses of synthetic miR-34a mimic or negative control miRNA formulated in lipid-based transfection reagent at 3-day intervals for 15 days (Supplementary Figure 1b). Whereas the tumors in the control group grew rapidly in this time frame, a 95% reduction in tumor growth was observed in the miR-34a-treated group (Figure 1a; Supplementary Figure 1c), which was independent of the lipofection reagent used (Supplementary Figures 2a-d). The difference in tumor size between the treatment groups was evident macroscopically (Figure 1b), and was reflected in significantly different tumor weights at the study endpoint (Figure 1c). Whereas miR-34a was hardly detectable in the tumors of the control group, its levels were strongly increased in the miR-34a-treated group (Figure 1d). The difference in miR-34a levels was not due to miRNA accumulation in the interstitial space but rather reflected its uptake into the tumor cells as documented in single cell suspensions after culturing ex vivo (Supplementary Figure 2e). Intratumoral delivery of synthetic miR-34a was also effective in preventing growth of tumors that were ~10-fold larger at treatment onset (Supplementary Figures 3a-d). An extension of the treatment to a total of 20 days in an independent experiment resulted in similar statistically significant differences in tumor volume and weight (Supplementary Figures 3e–h).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Intratumoral delivery of liposome-formulated miR-34a induces apoptosis and blocks DLBCL growth in vivo. NOD/SCID/IL2Rγ/ mice were subcutaneously inoculated in both flanks with 1 × 107 U2932 cells. Palpable tumors were locally injected every 3 days with either 12.5μg miR-34a (n=10) or 12.5μg scrambled negative control (NC) miRNA (n=10) for a period of 15 days. (a) Mean tumor volumes as recorded every 3 days. (b) Representative photograph taken at the study endpoint of xenograft-bearing mice treated with either NC miRNA (left) or miR-34a (right). (c) Scatter plot showing the tumor weights of the NC- and miR-34a-treated tumors at the study endpoint. (d) Quantitative real time PCR analysis of miR-34a levels of all tumors shown in (a and c); expression was normalized to U6 snRNA. (e and f) Representative micrographs taken at × 40 and × 320 (inset) magnification (e) and the overall quantification (f) of apoptotic and preapoptotic cells in the tumors shown in (a and c), as assessed by immunohistochemical staining for activated caspase-3. Note that two NC tumors were not available for staining. Black and white arrows point to early apoptotic cells (which are morphologically indistinguishable from live tumor cells, but caspase-3-positive) and late apoptotic cells (exhibiting strong nuclear caspase-3 staining and cytoplasmic condensation), respectively. Data represent mean±s.e.m.; horizontal bars indicate means. P values were obtained using Student's t-test. *P<0.05; **P<0.01; ***P<0.001. Data is representative of three independent experiments.

Full figure and legend (227K)

To investigate the mechanism underlying the tumor-suppressive effects of miR-34a replacement in vivo, we compared proliferation and apoptosis parameters in the two treatment groups. Explanted cells from miR-34a-treated tumors proliferated only marginally less than control cells as determined by [3H] thymidine incorporation (Supplementary Figure 4). In contrast, immunohistochemical staining for activated caspase-3 revealed significant differences in the fraction of early as well as late apoptotic cells in the tumor mass (Figures 1e and f). Taken together, the results provide evidence that DLBCL may be sensitive to a therapeutic strategy aimed at reintroducing miR-34a due to the strong proapoptotic properties of the miRNA.

Based on the encouraging results of the intratumoral treatments, we formulated synthetic miR-34a mimic or control miRNA in neutral lipid emulsion (NLE)9 and examined the therapeutic benefit of repeated systemic delivery. NOD/SCID/IL2Rγ/ mice implanted with U2932 cells were subjected to intravenous treatments once their tumors were ~50mm3 (Supplementary Figure 5a), and monitored closely with respect to tumor volumes and possible side effects for the duration of the treatment. None of the treated mice exhibited adverse symptoms (that is, weight loss, changes in feeding or grooming behavior) or developed tissue abnormalities that would have been evident at necropsy. A strong reduction in tumor growth by 76% on average was observed in mice receiving NLE-formulated miR-34a mimic compared with the mice in the control group (Figure 2a, Supplementary Figure 5b), which was again reflected in significantly different tumor weights at the study endpoint (Figure 2b). miR-34a-treated tumors expressed elevated levels of miR-34a and exhibited significantly higher apoptosis rates than the control tumors (Figures 2c-e).

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Systemic administration of lipid emulsion-formulated miR-34a suppresses growth of DLBCL in vivo. U2932 xenografts were generated in both flanks of NOD/SCID/IL2Rγ/ mice as described in Figure 1. Once tumors were palpable, mice were intravenously injected every 2 days with either 20μg of miR-34a (n=7) or negative control oligo (NC; n=7) formulated in neutral lipid emulsion for a total of 12 days. The last injection was performed 10min before the study endpoint. (a) Mean tumor volumes as recorded every 2 days. (b) Tumor weights of the NC and miR-34a-treated xenografts. (c) miR-34a levels as determined by quantitative real-time PCR of all tumors shown in a and b, normalized to U6 snRNA. (d and e) Representative micrographs taken at × 40 and × 320 (inset) magnification (d) and the overall quantification (e) of apoptotic and preapoptotic cells as assessed by immunohistochemical staining for activated caspase-3 of all tumors shown in ac. (f) Apoptotic cells as measured by annexin-V-binding assay 48h post-electroporation of U2932 cells with either scrambled-negative control miRNA (NC miRNA), mir-34a, scrambled negative control siRNA (NC siRNA) or FOXP1 siRNA. White bars represent samples that were simultaneously treated with z-VAD. (g) Western blotting for activated caspase-3, FOXP1 and actin of samples treated as described in f. Data are presented as mean±s.e.m.; horizontal bars indicate means. P values were obtained using Student's t-test. *P<0.05; **P<0.01; ***P<0.001. Results are representative of two (ae) to four (f and g) independent experiments.

Full figure and legend (261K)

To directly examine the proapoptotic effects of miR-34a, we introduced miR-34a into cultured U2932 cells by electroporation and quantified apoptotic cells by annexin-V-binding assay (Figure 2f). Annexin-V+ cells were significantly more abundant in cultures that had been electroporated with miR-34a relative to control miRNA (Figure 2f). The differential induction of apoptosis by miR-34a, but not control miRNA, was further reflected by western blotting for activated caspase-3 (Figure 2g). The effects of miR-34a were phenocopied to some extent by the siRNA-mediated knockdown of the miR-34a target FoxP1 (Figures 2f and g), suggesting that the proapoptotic effects of miR-34a are exerted, at least in part, through downregulation of this hematopoietic transcription factor and oncoprotein. However, the involvement of other miR-34a targets is likely, and off-target effects of this miRNA also cannot be excluded on the basis of our experiments. Addition of the pan-caspase inhibitor z-VAD prevented apoptosis induction by both miR-34a- and FoxP1-specific siRNA (Figure 2f). Interestingly, miR-34a delivery (and siRNA-mediated FoxP1 knockdown) also efficiently induced apoptosis in another DLBCL cell line lacking miR-34a expression, SUDHL6 (Supplementary Figures 6a and 1a), but had hardly any discernible effect in a DLBCL cell line, SUDHL7, expressing relatively normal levels of miR-34a (Supplementary Figures 6b and 1a).

In summary, the results presented here illustrate how miRNAs could be harnessed in the future for the treatment of aggressive lymphoma for which other targeted treatment modalities are lacking. Suitable patient cohorts may be identified based on their lack of tumor cell-intrinsic miR-34a expression in conjunction with overexpression of Myc, either due to chromosomal rearrangements involving the MYC locus10 or due to non-genetic events.11 miR-34a promoter methylation is common in DLBCL6 and, together with FoxP1 over-expression, could serve as another easily assessable indication for successful miR-34a replacement therapy.6, 8 Side effects of miR-34a treatment are projected to be minimal due to normal expression of the miRNA in non-tumor tissues.12 We have attributed the tumor-suppressive effects of miR-34a to its anti-apoptotic properties in vitro and in vivo, and to the specific knockdown of FoxP1. Further elucidation of the mechanism of miR-34a/siFoxP1-driven apoptosis remains a challenge for future experiments.

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Conflict of interest

AGB is an employee of Mirna Therapeutics, Inc., which develops miRNA-based therapeutics. All the other authors declare no conflict of interest.

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References

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Acknowledgements

We would like to thank Stephan Dirnhofer and Sergio Cogliatti for helpful discussions. This study was funded by grants from the Swiss Cancer League and the Zurich University Research Priority Program in Systems Biology to AM.

Supplementary Information accompanies the paper on the Leukemia website