Akt inhibition improves long‐term tumour control following radiotherapy by altering the microenvironment

Abstract Radiotherapy is an important anti‐cancer treatment, but tumour recurrence remains a significant clinical problem. In an effort to improve outcomes further, targeted anti‐cancer drugs are being tested in combination with radiotherapy. Here, we have studied the effects of Akt inhibition with AZD5363. AZD5363 administered as an adjuvant after radiotherapy to FaDu and PE/CA PJ34 tumours leads to long‐term tumour control, which appears to be secondary to effects on the irradiated tumour microenvironment. AZD5363 reduces the downstream effectors VEGF and HIF‐1α, but has no effect on tumour vascularity or oxygenation, or on tumour control, when administered prior to radiotherapy. In contrast, AZD5363 given after radiotherapy is associated with marked reductions in tumour vascular density, a decrease in the influx of CD11b+ myeloid cells and a failure of tumour regrowth. In addition, AZD5363 is shown to inhibit the proportion of proliferating tumour vascular endothelial cells in vivo, which may contribute to improved tumour control with adjuvant treatment. These new insights provide promise to improve outcomes with the addition of AZD5363 as an adjuvant therapy following radiotherapy.

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Referee #1 (Remarks):
This is an excellent piece of work by a strong radiobiology laboratory and addresses a unique aspect of RT-DRUG combinations when targeting AKT signaling: a pathway associated with poor local control in HEENT tumours.
The authors show convincingly that the AKT-inhibitor AZD5363 (AKTi) was much more effective as an adjuvant treatment than when given as a neoadjuvant to RT in two HEENT models (FaDu and PE/CA PJ34); importantly-in some animals-this led to complete local control. This effect was independent of the effects on the intrinisic radiosensitivity of cell lines in vitro. The adjuvant effect was associated with altered density, tumour vasculature endothelial function and correlated with an influx of CD11b+ myeloid cells in vivo.
Overall, the study is well conducted in two independent HEENT models with similar findings.
I have a few comments and suggestions that would make the publication's conclusions stronger: 1) Are there effects of AKT signaling on the cell cycle phase at the time of irradiation -could the sensitization in vivo also be due to altered cell cycle phase after prolonged in vivo treatments ? Have the authors completed ex vivo clonogenic survival curves with 6 or 10Gy to show radiosensivity in vivo is not affected ?
2) Can the authors speculate or have data on the results if fractionated radiotherapy was used and the effects of increasing hypoxia on these intra-tumoural sensitivity with the agent ?
3) What biomarkers should be used for the PD approaches to the use of this agent in clinical trials ? Which patients might or might not benefit and how would one test a priori or intra-therapy for its continued adjuvant use ? This is important to show the translational aspects of the work. 4) What toxicity -if any-did the RT-drug combo elicit, or could elicit, with results of this study using the combined modality approach ? Please comment on the therapeutic ratio and data pertaining to it if available.
Referee #2 (Remarks): Searle et. al. provide an interesting study that systematically evaluates how blockade of Akt with ADZ5363 and treatment with ionizing radiation is not effective on tumor cells but alters the function of the cells in the tumor microenvironment. There are some points of clarification and interpretations that need to be address to support the claims.
1. For the analysis of endothelial cells with ADZ5363 and IR the authors should consider constructing an isobologram to assess if there is an additive or synergistic effect. 2. While the data on ADZ5363 on tumor cells shows that it inhibits Akt in tumor cells alone, it is unclear if this compound with IR is still effective as no western blots were provided. 3. Is it possible that a subpopulation of tumor cells with IR+ADZ5363 undergo senescence, which would explain the decrease in VEGF? 4. The Figure S1 legend does not reflect the text on page 13 top paragraph. 5. It was not clear in Figure S5A and B if there was IR treatment alone. It was not clearly marked in the Figure. 6. In Figure 6 the data shows CD31 staining for vessels but it is unclear if the analysis was to measure all vessels. IT might be important to distinguish between mature vessels versus immature vessels. 7. The author suggesting that endothelial cells are most sensitive to ADZ5363 and IR. Could the author substantiate these finding by providing evidence that circulating endothelial progenitor cells in the animal are sensitive to the ADZ5363, IR or ADZ5363 and IR. This would provide evidence that source of the endothelial cells are from the bone marrow versus the mature vasculature. Many thanks for forwarding the helpful and insightful comments from the reviewers. We are grateful for the opportunity to revise and enhance the manuscript in light of this feedback and suggestions. We have done our best to address the specific comments made by the reviewers alongside the changes made and these are outlined point by point below.
Referee #1: 1) Are there effects of AKT signaling on the cell cycle phase at the time of irradiation -could the sensitization in vivo also be due to altered cell cycle phase after prolonged in vivo treatments? Have the authors completed ex vivo clonogenic survival curves with 6 or 10Gy to show radiosensitivity in vivo is not affected? This is a potentially important point and we agree that Akt inhibition with AZD5363 could have an effect on cell cycle at the time of irradiation. We have investigated this in FaDu cells and did not originally include our data as we found no evidence of an effect of AZD5363 on cell cycle measured by flow cytometric analysis with propidium iodide. In the light of the reviewers questions this data has been added into Appendix figure S3J and in the manuscript (page 5, para 4). This data demonstrates that non-toxic concentrations of AZD5363 have no effect on cell cycle, either alone, or 24hrs after 4 Gy irradiation. This is in keeping with our hypothesis that the impact of AZD5363 on the response of tumours to radiotherapy does not result from a direct effect of cellular radiosensitivity, but is due to an impact on the tumour microenvironment. These results are consistent with with published results on cell cycle analysis of prostate cancer cell lines where in PC3/DU145 cells, a 10 µM concentration of AZD5363 had no cytotoxic effect and this was accompanied by no effects on cell cycle (Lamoureux et al, 2013). The MTT assays we performed revealed FaDu to be an insensitive cell line, and as such our results are in keeping with this publication. Our exhaustive in vitro experiments clearly demonstrated no impact on intrinsic radiosensitivity. This, when coupled with a lack of cell cycle effect, meant that while we did consider the possibility of assessing ex vivo clonogenic survival early on in our program, we rationalised to focus on the microenvironment.

2) Can the authors speculate or have data on the results if fractionated radiotherapy was used and the effects of increasing hypoxia on these intra-tumoural sensitivity with the agent?
Whether AZD5363 will improve long-term tumour control after fractionated radiotherapy is indeed an important question and will be the subject of future planned work. However, this work is beyond the scope of this proof of principle initial study. We would speculate that as the mechanism by which AZD5363 improves control after radiotherapy is at least, in part, derived from the effects of AZD5363 of the oxygen independent regulation of HIF, that we are optimistic of an effect following fractionated radiotherapy. We have addressed this with additional comments in the discussion (page 17, para 2).

3) What biomarkers should be used for the PD approaches to the use of this agent in clinical trials? Which patients might or might not benefit and how would one test a priori or intratherapy for its continued adjuvant use? This is important to show the translational aspects of the work.
Developing suitable biomarkers is also an important part of our future work and in informing the translation of this work into early phase clinical trials. In the phase I trial investigating AZD5363 in advanced solid tumours pPRAS40 inhibition in the hair follicle was successfully used to demonstrate targeting of the Akt pathway in patients given AZD5363 (Banerji et al, 2013). In addition, Akt1 and PIK3CA gene mutations were found to be associated with the largest responses to AZD5363 in terms of tumour reduction. However, both of these approaches might be expected to only inform to a limited degree which patients might benefit from a combination approach of AZD5363 with radiotherapy. As both mouse models were relatively insensitive to AZD5363 monotherapy, it appears that monotherapy efficacy is not required for the combination of AZD5363 and radiotherapy to produce successful long term tumour control. Whilst on-target Akt inhibition via examination of the hair follicle may be an a priori test, other potential biomarkers will need investigation and validation in relation to combination efficacy. We consider that potential biomarkers may include vascular endothelial cell proliferation and high HIF-mediated gene expression in the pre-treatment biopsy sample. Intra-therapy-imaging of vascular function (DCE-MRI) would also be a potential biomarker for future investigation. We have added a comment on these approaches into our discussion (page 17, para 3).

4) What toxicity -if any-did the RT-drug combo elicit, or could elicit, with results of this study using the combined modality approach? Please comment on the therapeutic ratio and data pertaining to it if available.
There were no discernible differences in toxicity between the combination, single treatment and control arms in either experiment. Mouse weight data has now been included in supplementary figure S4A. The adverse effects seen in the Phase I trial with AZD5363 included hyperglycaemia and diarrhoea and appeared dose related. The recommended phase II dose for AZD5363 is 320 mg BD for continuous dosing. The dosing of 400 mg BD is approximately equivalent to 100 mg/kg BD preclinical exposure (in the mouse), indicating equivalent drug doses to the 50mg/kg BD pre-clinical exposure used in our work are achievable. The radiation dose used in our work was not high enough to cause any effect on tumour growth alone, therefore in our studies the benefit of AZD5363 is achieved at no "cost" with respect to normal tissue toxicity. Although this infers high therapeutic ratio, this is a very large claim to make from a xenograft model in a short term experiment. Given our proposal that AZD5363 should be given as an adjuvant to RT thereby negating potential for exacerbating effects during RT, we do not anticipate that combination toxicity should be greater that the known toxicities of the individual treatment modalities. However, this will need to be further assessed in future early phase clinical trials of the combination. We have now included further discussion of the dosing of AZD5363 used in our work, increasing the therapeutic ratio and how this might translate into future combination clinical trial design (page 16, para 3).

Referee #2: 1. For the analysis of endothelial cells with ADZ5363 and IR the authors should consider constructing an isobologram to assess if there is an additive or synergistic effect.
We have previously demonstrated that treating endothelial cells with AZD5363 alone inhibits the proliferation of vascular endothelial cells, using a BrdU assay. In light of the reviewer's insightful comments we have conducted some further experiments to investigate whether there is a greater than additive effect from AZD5363 on the proliferation of HUVEC cells when treated with 6 Gy irradiation. These additional experiments have been included (Extended View Figure EV3, and in the manuscript page 12, para 1) and demonstrate that there was no additive increase in cell killing of the HUVEC endothelial cells regardless of whether AZDD5363 was given before and after the RT. Given this lack of additive cell kill we have not considered drawing an isobologram.

While the data on ADZ5363 on tumor cells shows that it inhibits Akt in tumor cells alone, it is unclear if this compound with IR is still effective as no western blots were provided.
As requested, we have now included a western blot demonstrating the effect of AZD5363 on irradiated FaDu cells ( Figure 1G and manuscript page 6, para 1).

Is it possible that a subpopulation of tumor cells with IR+ADZ5363 undergo senescence, which would explain the decrease in VEGF?
The referee makes an interesting point and whilst it is possible that a subpopulation of tumour cells undergo senescence with the combination of RT+AZD5363, it would appear that senescence is not required in order to see a decrease in VEGF. The VEGF data depicted in Figure 5 demonstrates Human VEGF is reduced in FaDu tumours from mice treated with AZD5363 or vehicle alone. In this model, AZD5363 has no significant effect on tumour growth at the dose used (50 mg/kg BD), and our interpretation is that significant cellular senescence is unlikely to have occurred. In addition, in Figure 6E we demonstrate that Human Ki67 is not reduced in FaDu tumours with treatment with AZD5363 alone. This material is from the same tumours used to generate the VEGF data, again indicating senescence is not required for a reduction in VEGF. A comment on this has now been made in the paper (page 14, para 1). Figure S1 legend does not reflect the text on page 13 top paragraph. We wish to thank the referee was noticing this error and have made the correction to the Figure reference. Figure S5A and B if there was IR treatment alone. It was not clearly marked in the Figure. We wish to clarify that there is no data for an RT alone treated mouse. Our intention was to compare combination treatment with RT alone, however the tumour on the RT alone treated mouse rapidly outgrew the tumour window necessitating culling of that animal only 3 days after treatment began. As such, this experiment was hypothesis generating, allowing us to focus our studies on the tumour vasculature to the 7 days post RT time point, using a different method of analysis; CD31 staining (immunohistochemistry). This has been made clearer in the figure legend.

In Figure 6 the data shows CD31 staining for vessels but it is unclear if the analysis was to measure all vessels. IT might be important to distinguish between mature vessels versus immature vessels.
In our analysis of blood vessels we felt it was important to consider the effect of AZD5363 post radiotherapy on all vessels, both mature and immature. As such we chose to use CD31 as it can be expected to stain all vessels (both mature and immature) rather than CD34, which tends to stain more immature vessels only. This was so as not to miss an effect on established, rather than just developing vasculature. This has been clarified in the manuscript (page 11, para 2) and the mislabelling in Figure 4A has been corrected.
7. The author suggesting that endothelial cells are most sensitive to ADZ5363 and IR. Could the author substantiate these finding by providing evidence that circulating endothelial progenitor cells in the animal are sensitive to the ADZ5363, IR or ADZ5363 and IR. This would provide evidence that source of the endothelial cells are from the bone marrow versus the mature vasculature.
The discussion in our original submitted manuscript outlined our interpretation of our results that whilst AZD5363 reduces the proliferative rate of vascular endothelial cells, it was the effects of AZD5363 in the wider tumour microenvironment that resulted in enhanced tumour control after radiotherapy. We did not present data to suggest a direct effect on the radiosensitivity of vascular endothelial cells with the addition of AZD5363. However, following the helpful reviewers comments, we performed additional experiments and the data from these experiments is included (supplementary Figure S7) demonstrating that there is not a greater than additive effect on HUVEC cell proliferation when AZD5363 and radiation are combined, suggesting endothelial cells are not especially sensitive to the combination. We hope that our revisions will satisfy the reviewers. We have also reformatted the manuscript in order to meet the requirements as specified in the author guidelines. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine.
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***** Reviewer's comments ***** Referee #2 (Remarks for Author): Reviewer 1: The authors were responsive to the previous concerns raised. However, the data related to ADZ5363 cell cycle appears to show differences before irradiation, if the cell cycle phases (s and G2) are examined closely. It does not appear to have had stats performed on the cell cycle data either.
Reviewer 2: Overall the authors responded to the points raised. Many thanks for agreeing to accept our manuscript for publication pending satisfactory compliance with the reviewer's final requests.
Reviewer 1: The authors were responsive to the previous concerns raised. However, the data related to ADZ5363 cell cycle appears to show differences before irradiation, if the cell cycle phases (s and G2) are examined closely. It does not appear to have had stats performed on the cell cycle data either.
Although we appreciate that when looking at the figure there is the suggestion of possible differences, statistical analysis did not show any difference when AZD5363 treated cells were compared with their respective irradiated and mock-irradiated controls. The type of analysis performed was the Kruskal-Wallis test followed by Dunn's multiple comparisons test. We apologise for not having included this information and this has now been added on p24 on the manuscript. We have also added labels to the figure to indicate the comparisons made. We hope that these changes will meet with your approval. Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.
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Mouse numbers were determined by the calculation of group sizes needed to obtain a biologically relevant 40% change from control (assuming the significance level is set at 5%) with a power of > 80%, based on previous growth data using InVivostat software (www.invivostat.co.uk).
Mouse numbers were determined by the calculation of group sizes needed to obtain a biologically relevant 40% change from control (assuming the significance level is set at 5%) with a power of > 80%, based on previous growth data using InVivostat software (www.invivostat.co.uk).
n/a Mice bearing tumours measuring 100 mm3 in size were randomized into AZD5363 (50 mg/kg, PO, BD) alone, RT alone, vehicle alone and combination treatment groups.
Mice bearing tumours measuring 100 mm3 in size were randomized into AZD5363 (50 mg/kg, PO, BD) alone, RT alone, vehicle alone and combination treatment groups.
Mice were randomised as tumours reached starting tumour volume. The investigator was not blinded as to the treatment group of the mice.
No blinding steps were undertaken Yes Yes. Tumour volumes were log transformed to stabilise variance and remove size dependency; variance was unequal between groups as it increases with tumour volume.
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