“Pharmacologic targeting of NR4A1 and NR4A2 to activate glioblastoma treatment response” - Resistance to standard of care (SOC) temozolomide (TMZ) and radiation treatment (XRT) limits survival of the most common adult brain cancer, glioblastoma (GBM), to 12-18 months. Despite promising results in other cancers, robust immunosuppression in GBM has also limited the responses to immune checkpoint inhibitors (ICIs). Reciprocal interactions in the GBM tumor microenvironment (TME) between mesenchymal changes and immunosuppression enhance resistance to chemoradiation and immunotherapy, respectively. Therefore, a compelling therapeutic strategy for GBM is to concurrently reprogram the mesenchymal and immune suppressive TMEs and potentiate ICI and SOC therapy. The NR4A1 and NR4A2 orphan nuclear receptors are compelling targets to achieve this goal. We synthesized a series of novel bis-indole–derived ligands (CDIMs) with potent dual antagonism of NR4A1 and NR4A2 and negligible in vivo toxicity, of which six demonstrated nM range KDs for both receptors. Genetic based NR4A 1 and 2 loss of function strongly activates anti-cancer immune responses by reversing T-cell exhaustion which underlies poor ICI responses in GBM. In addition, NR4A1/2 promote EMT in other cancers which contributes to chemoradiation resistance. Finally, high levels of NR4A1/2 expression in GBM are strongly associated with decreased patient survival after conventional treatments. Therefore, we propose to test the hypothesis that CDIM inhibition of NR4A1/2 reprograms the GBM TME and potentiates ICI and TMZ/XRT responses. Further NR4A1/2 upregulates PD-L1 and TWISt1, key regulators of GBM immune suppression and mesenchymal phenotypes, respectively. Preliminary studies demonstrated that CDIMs inhibit PD-L1 and TWIST1 expression, reverse immune suppressive myeloid and T cell phenotypes and prolong survival of experimental GBMs. We will test our hypothesis by i) identifying lead dual NR4A1/2 CDIM compounds based on inhibition of malignant and mesenchymal GBM cell properties (proliferation, self-renewal, invasion) and reversal of dysfunctional T cell phenotypes in vivo (Aim 1), ii) establishing the functional impact of NR4A1/2 CDIMs to reprogram the immune suppressive and mesenchymal TME (Aim 2) and iii) quantifying the effects of NR4A1/2 CDIMs to potentiate ICI and SOC responses in mouse GBM syngeneic models and define the role of PD-L1 and TWIST1 in their mechanisms of action (Aim 3). Quantification of potency, pharmacokinetics and preclinical anti-tumor activity alone and in combination with to standard of care TMZ-XRT and ICIs is expected to identify candidate CDIMs for consideration in future clinical trials. Through application of powerful bioinformatic tools that model the cancer TME through integration of scRNAseq, cyTOF and IMC data, these studies are expected to shed new light on novel mechanisms by which mesenchymal and immune suppressive TMEs interact to coordinately drive treatment resistance in GBM through NR4A1/2. To achieve our goals, we assembled a unique multidisciplinary team with expertise in medicinal chemistry, neuro-oncology, GBM biology and preclinical modeling, and immuno-oncology.
