Pharmacologic Inhibitors of STAT3 or BCL6 Transcriptional Function Sensitize Lymphoma Cells to the Novel PD-1 Cis-Targeted PD1-IL2v Immunocytokine in a Murine Model

Although immune-based therapies like immune checkpoint inhibitors or CAR-T cells have led to dramatic advances in treating relapsed and refractory lymphomas, most patients still do not derive a durable response. Lymphoma cells, while being immunogenic, can have intrinsic mechanisms of resistance to immune-based killing and may also establish an immuno-suppressive microenvironment in which immune effector cells have diminished activity. This immune resistance may be a direct result of the inappropriate and constitutive activation of transcription factors whose physiologic function is to promote survival and resistance to immune-based killing. Two transcription factors that play such a role in lymphomas are STAT3 and BCL6. We therefore wished to test the hypothesis that drugs that block the transcriptional function of these proteins would enhance the efficacy of an immune-based therapy.

We chose to use the A20 murine lymphoma system, which grows as an aggressive tumor in syngeneic Balb/c mice, and is generally resistant to immune checkpoint inhibitors such as PD-1 antagonists. A20 cells are characterized by constitutive STAT3 activation and BCL6 expression. To enhance the translational relevance of this study, we used inhibitors of STAT3 or BCL6 transcriptional function that could rapidly be introduced into clinical trials. We identified drugs that inhibit the transcriptional effects of these proteins based on a computational search of drugs that are known to be safe in humans, and which lead to gene expression changes opposite to that driven by each transcription factor. Using this approach, we identified atovaquone as a STAT3 inhibitor and disulfiram as a BCL6 inhibitor. The activity of each drug was then confirmed by in vitro testing. Furthermore, the resultant changes in gene expression in A20 cells would be predicted to make these cells more immunogenic. To stimulate the endogenous anti-tumor immune response, we utilized a novel murine specific PD-1 cis-targeted PD1-IL2v immunocytokine. muPD1-IL2v is, a fusion protein consisting of a PD-1 antibody and a C-terminally fused variant form of the cytokine IL-2 (IL2v) that does not bind to CD25. CD25 is the alpha chain that confers high affinity to the IL-2 receptor and which is highly expressed on immunosuppressive T regulatory cells (Tregs). This design enables muPD1-IL2v to preferentially activate PD-1 expressing antigen specific T effector cells by removing the competitive advantage of immunosuppressive T regulatory cells. At the same time, the PD-1 targeting allows the preferential delivery of IL-2v to PD-1⁺ tumor specific lymphocytes while bypassing the binding to Tregs.

We then tested the activity of atovaquone or disulfiram in combination with muPD1-IL2v in an immunocompetent model with A20 lymphoma cells. As a single agent, muPD1-IL2v had a modest effect in delaying the growth of these tumors. Similarly, atovaquone or disulfiram each showed only a small change in the rate of growth of A20 tumors. However, when used in combination with muPD1-IL2v, each of these transcriptional inhibitors led to a significant fraction of animals having complete and long term regression of these tumors. Furthermore, tumors resected from animals treated with atovaquone showed evidence of on-target inhibition of STAT3 function, and tumors resected from animals treated with disulfiram showed evidence for on-target effects on BCL6 target gene expression.

These findings suggest that inhibition of oncogenic transcription factors like STAT3 and BCL6, in conjunction with novel immune-targeted therapeutics like PD1-IL2v, have the potential for long term disease control in lymphomas.

This research collaboration was supported through the imCORE network.

Herter:Roche Glycart AG: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Codarri Deak:Roche: Current Employment, Current equity holder in publicly-traded company. Klein:Roche Glycart AG: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Atovaquone (approved as an anti-parasitic drug), used as an inhibitor of STAT3. Disulfiram (approved for use in alcohol dependence), used as an inhibitor of BCL6.