Dual kinase inhibitor for EGFR mutants and ErbB2 limit breast cancer.

Mutations in the epidermal growth factor receptor (EGFR) have been found in more than 10% of non-small cell lung cancer (NSCLC) patients in North America. The vast majority of these differences are L858R point mutations in Exon 21. Currently, monoclonal antibodies directed against the extracellular domain of EGFR or small molecule/tyrosine kinase inhibitors (TKI) are the stalwarts of NSCLC therapy. Resistance, however, gradually develops because of the T790 mutation towards first and second generation TKIs. The third generation TKI AZD9291 (Osimertinib) has a high affinity for both activating and the acquired resistant mutation (T790 M) in EGFR, with a low affinity towards wild-type EGFR. Recent research, however, suggests that the EGFR (C797S) mutation in the tyrosine kinase domain is a likely cause of resistance to AZD9291. Another significant transformation mechanism associated with this resistance is erbB2 amplification. Our laboratory has developed a small kinase inhibitor, ER121 (MW: ∼500), that inhibits the erbB2/HER2 tyrosine kinases in addition to the EGFR C797S mutations. We have identified a TKI, ER121 targeting the mutant EGFR(T790 M). Using in vitro and in vivo models, examined the efficacy of ER121 on mutant EGFR cell lines. This has enabled us to establish that ER121 is well tolerated when administered orally and produces significant inhibitory activity against human cancers generated by mutant EGFR and amplified ErbB2.

PRMT5 Associates With the FOXP3 Homomer and When Disabled Enhances Targeted p185erbB2/neu Tumor Immunotherapy.

Regulatory T cells (Tregs) are a subpopulation of T cells that are specialized in suppressing immune responses. Here we show that the arginine methyl transferase protein PRMT5 can complex with FOXP3 transcription factors in Tregs. Mice with conditional knock out (cKO) of PRMT5 expression in Tregs develop severe scurfy-like autoimmunity. In these PRMT5 cKO mice, the spleen has reduced numbers of Tregs, but normal numbers of Tregs are found in the peripheral lymph nodes. These peripheral Tregs that lack PRMT5, however, display a limited suppressive function. Mass spectrometric analysis showed that FOXP3 can be di-methylated at positions R27, R51, and R146. A point mutation of Arginine (R) 51 to Lysine (K) led to defective suppressive functions in human CD4 T cells. Pharmacological inhibition of PRMT5 by DS-437 also reduced human Treg functions and inhibited the methylation of FOXP3. In addition, DS-437 significantly enhanced the anti-tumor effects of anti-erbB2/neu monoclonal antibody targeted therapy in Balb/c mice bearing CT26Her2 tumors by inhibiting Treg function and induction of tumor immunity. Controlling PRMT5 activity is a promising strategy for cancer therapy in situations where host immunity against tumors is attenuated in a FOXP3 dependent manner.

A targeted immunotherapy approach for HER2/neu transformed tumors by coupling an engineered effector domain with interferon-γ.

Despite substantial clinical progress with targeted therapies, current antibody-based approaches have limited efficacy at controlling HER2/neu-positive breast cancers, especially in the absence of chemotherapies. Previously, we showed that the combination of IFNγ and anti-HER2/neu antibody synergistically reduces tumor growth in an in vivo implanted mammary tumor model. Here, we report a recombinant approach to produce an anti-HER2/neu scFv and IFNγ fusion protein using an engineered effector domain (EED) scaffold. The new molecule induces in vitro apoptosis in an IFNγ receptor-dependent manner. At a very low dose in the in vivo xenografted tumor models, the new EED-IFNγ fusion protein demonstrates superior activity over the anti-HER2/neu antibody and is even active on tumors that are resistant to anti-HER2/neu antibody therapy. Examination of tumor infiltrated macrophages and lymphocytes reveals that the fusion protein can induce changes in tumor microenvironment to support immune reactivity against tumors. Our studies have defined a targeted immunotherapy approach for the treatment of cancers.

Disabling of the erbB Pathway Followed by IFN-γ Modifies Phenotype and Enhances Genotoxic Eradication of Breast Tumors.

Reversion of the malignant phenotype of erbB2-transformed cells can be driven by anti-erbB2/neu monoclonal antibodies (mAbs), which disrupt the receptor's kinase activity. We examined the biologic effects of IFN-γ alone or after anti-erbB2/neu mAb treatment of erbB2-positive cells. IFN-γ had no effect on its own. Treatment of the tumors with anti-erbB2/neu mAbs followed by IFN-γ led to dramatic inhibition of tumor growth in vitro and in vivo with minimal mAb dosing. Sequential therapy enhanced the effects of chemotherapy. Moreover, IFN-γ with mAb treatment of mice with IFNγR knockdown tumors did not demonstrate marked synergistic eradication effects, indicating an unexpected role of IFN-γ on the tumor itself. Additionally, mAb and IFN-γ treatment also induced immune host responses that enhanced tumor eradication. Biochemical analyses identified loss of Snail expression in tumor cells, reflecting diminution of tumor-stem-cell-like properties as a consequence of altered activity of GSK3-ß and KLF molecules.

Dynamic interactions between TIP60 and p300 regulate FOXP3 function through a structural switch defined by a single lysine on TIP60.

The human FOXP3 molecule is an oligomeric transcriptional factor able to mediate activities that characterize T regulatory cells, a class of lymphocytes central to the regulation of immune responses. The activity of FOXP3 is regulated at the posttranslational level, in part by two histone acetyltransferases (HATs): TIP60 and p300. TIP60 and p300 work cooperatively to regulate FOXP3 activity. Initially, p300 and TIP60 interactions lead to the activation of TIP60 and facilitate acetylation of K327 of TIP60, which functions as a molecular switch to allow TIP60 to change binding partners. Subsequently, p300 is released from this complex, and TIP60 interacts with and acetylates FOXP3. Maximal induction of FOXP3 activities is observed when both p300 and TIP60 are able to undergo cooperative interactions. Conditional knockout of TIP60 in Treg cells significantly decreases the Treg population in the peripheral immune organs, leading to a scurfy-like fatal autoimmune disease.