Alfonso Bellacosa, MD, MHS

Alfonso Bellacosa, MD, MHS

Fox Chase Cancer Center

Research Project:
Improving Response Rates of Immunotherapy for Lung Cancer

Grant Awarded:

  • Lung Cancer Discovery Award

Research Topics:

  • basic biologic mechanisms
  • combination therapies experimental therapeutics
  • gene expression transcription
  • immunology immunotherapy

Research Disease:

  • lung cancer

While my laboratory stayed open during the pandemic, we faced difficulties related to availability of supplies and reagents, and decreased facility access, as facilities were working with reduced personnel while facing demands from multiple laboratories. Immune checkpoint blockade (ICB) with anti-PD1, anti-PD-L1 and anti-CTLA4 antibodies is an exciting and effective new treatment for advanced lung cancer (LC). Unfortunately, only about 40% of treatment naïve patients respond to ICB and only 20% can have long-term benefit. A significant need remains to render immunotherapy effective for a larger fraction of LC patients. By examining the TCGA lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) datasets, we found that a significant fraction (~20%) of LC cases exhibit prominent genome-wide DNA hypomethylation that correlates with upregulation of Thymine DNA Glycosylase (TDG) and Ten Eleven Translocation 1 (TET1), enzymes that work sequentially to demethylate methyl-CpG sites. Importantly, gene set variance analysis revealed an enrichment of oncogenic MYC target-related hallmarks and depletion of inflammation-related hallmarks in the highly hypomethylated group, raising the possibility that elevated TET1/TDG activity contributes to both oncogenic signaling and immunoevasion/resistance to ICB. Recently, we published two critical results: 1) TDG knockdown in melanoma cells leads to DNA hypermethylation and decreased proliferation; 2) TDG knockdown potently induces expression of cytokines and chemokines of the senescence-associated secretory phenotype, that are associated with the inflammatory/interferon response. Thus, we hypothesize that inhibiting TDG could be an innovative epigenetic approach to reprogram and correct the hypomethylated epigenome of LC, achieving a dual therapeutic benefit, via transcriptional repression of oncogenic activation and by rendering “cold,” refractory LC cases “hot” and susceptible to immunotherapy. We screened a virtual chemical library and identified a lead compound, MC1, with TDG inhibitory activity in vitro. This is the first known chemical inhibitor of TDG, and we are using it as a tool compound to test this novel epigenetic approach and for drug development. Despite the challenges of a pandemic year, we made significant progress in this project, as detailed below, obtaining data that strengthen our hypothesis and give us confidence for the goals we have set for the coming year. Our overall approach has been centered on developing animal models to maximize the clinical potential of this discovery: specifically, we want to develop a new transgenic mouse of hypomethylated lung cancer (Aim 1), and directly test our potent TDG inhibitor in combination with ICB for lung cancer treatment in syngeneic, immunocompetent mouse (Aim 2). For the studies in Aim 1, we devised a two-prong strategy, by engineering a “Super-eraser” mouse strain and taking advantage of our Tdg flox mice. First, we designed a targeting construct for overexpression, at the Rosa26 locus, of both TET1 and TDG, which not only is expected to induce more extensive DNA demethylation compared to single gene overexpression (hence the name “Super-eraser” of methylation marks), but will also simplify crosses to the KRASG12D p53-/- mice. Importantly, we decided to engineer two different degradation domains, with TDG fused to the afore-mentioned DHFR degradation domain regulatable by trimethoprim, and TET1 fused to FKBP degradation domain regulatable by Shield-1; thus, it will be possible to eliminate at will the expression of TET1 and TDG independently from each other and within the same mouse background, to assess the respective contributions of each protein to the LC phenotypes. Second, as an alternative and non-overlapping approach, we decided to use our Tdg flox mice. We initiated crosses of our conditional Tdg flox mice with the KRASLSL-G12D p53flox/flox mice available in the laboratory of our collaborator Dr. Hossein Borghaei (Fox Chase Cancer Center), to generate experimental Tdgflox/flox KRASLSL-G12D p53flox/flox mice, and control Tdg+/+ KRASLSL-G12D p53flox/flox mice. Upon delivery of Adeno-Cre by intratracheal intubation, there will be concomitant inactivation of Tdg and p53, and activation of KRAS G12D. While this mouse will not allow us to study the effects of acute TET-TDG inactivation as the Super-eraser mouse, it will still provide valuable information, regarding KRAS-p53-driven lung tumor formation/incidence/burden in the absence of Tdg. This in turn may provide additional validation of our hypothesis, in conjunction with the studies of the Super-eraser mouse. The purpose of Aim 2 is to evaluate the activity of TDG inhibitors in vivo (in KP1 syngeneic mice), in combination with anti-PD(L)1 immunotherapy. I am happy to report that in the past year, via a collaboration with Dr. Jian Jin, Mount Sinai Endowed Professor in Therapeutics Discovery, a leader in medicinal chemistry, we have identified more potent MC1 derivatives. In addition, the Institutional Animal Care and Use Committee has approved our animal protocol for dose escalation studies in mice for both MC1 and its derivatives. These studies will be promptly started in the next few weeks, as soon as our Organic Chemistry facility will prepare oral formulation of MC1 (in water, PBS or corn oil) for gavage, for which we have experience, or, alternatively, for intravenous or intraperitoneal administration. As soon as this facility will synthesize large-scale preparations of the potent MC1 derivatives, the animal studies can be extended to these compounds. Finally, In the interim of writing a more complete paper describing the TDG inhibitors, we have obtained data using infection of LC lines with lentivirus expressing shRNA against TDG, that TDG is a bona fide target in lung cancer, as its knockdown impairs viability and colony formation. We plan to submit a manuscript using these data before the summer. In summary, we are honored to have received the support of the American Lung Association for these studies. If these animal studies are successful, we plan to initiate a small Phase 1 clinical trial with TDG inhibitors, which could potentially be supported by the American Lung Association. We remain motivated to bring this new class of compounds to the clinic with the aim of eradicating deaths from lung cancer.

Update: We are studying the TET1-TDG DNA demethylase pathway in lung carcinogenesis. In the past year, we engineered a transgenic mouse that inducibly overexpresses TET1 and/or TDG along with lung-specific KRAS-G12D activation and p53 inactivation (Aim 1). In parallel, we are generating a mouse for simultaneous and inducible lung-specific activation of KRAS-G12D and inactivation of p53 and Tdg. We are moving forward with animal studies with our small-molecule TDG inhibitor. We now have IACUC approval to conduct dose escalation studies in mice for TDG inhibitors. This is the first step towards testing our potent TDG inhibitor in combination with immunotherapy (Aim 2).

Supported by the Mary Fuller Russell Fund

Page last updated: December 5, 2022

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