Small molecule inhibitors for cancer immunotherapy and associated biomarkers - the current status

Abstract

Following the success of cancer immunotherapy using large molecules against immune checkpoint inhibitors, the concept of using small molecules to interfere with intracellular negative regulators of anti-tumor immune responses has emerged in recent years. The main targets for small molecule drugs currently include enzymes of negative feedback loops in signaling pathways of immune cells and proteins that promote immunosuppressive signals within the tumor microenvironment. In the adaptive immune system, negative regulators of T cell receptor signaling (MAP4K1, DGKα/ζ, CBL-B, PTPN2, PTPN22, SHP1), co-receptor signaling (CBL-B) and cytokine signaling (PTPN2) have been preclinically validated as promising targets and initial clinical trials with small molecule inhibitors are underway. To enhance innate anti-tumor immune responses, inhibitory immunomodulation of cGAS/STING has been in the focus, and inhibitors of ENPP1 and TREX1 have reached the clinic. In addition, immunosuppressive signals via adenosine can be counteracted by CD39 and CD73 inhibition, while suppression via intratumoral immunosuppressive prostaglandin E can be targeted by EP2/EP4 antagonists. Here, we present the status of the most promising small molecule drug candidates for cancer immunotherapy, all residing relatively early in development, and the potential of relevant biomarkers.

Keywords: T-cell receptor signaling; adenosine; biomarker; cGAS/STING; cancer immunotherapy; immuno-oncology; small molecule inhibitors; tumor microenvironment.

Figure 1

Negative regulators of the TCR and associated immune pathways. An intricate network of negative regulators ensures that signals contributing to the activation of T cells are correctly terminated after a certain period of active signaling. In the case of the TCR, following receptor binding to MHC-bound ligands, receptor distal signaling is down-modulated through a negative feedback loop by PTPN2 and PTPN22 which dephosphorylate LCK. ZAP70 activity is controlled by SHP-2 which dephosphorylates this kinase and CBL-B which ubiquitinates ZAP70. In parallel, CBL-B directly ubiquitinates and inactivates the TCR itself. In addition to this function, CBL-B negatively impacts the CD28 costimulatory pathway by ubiquitinating the PI3K subunit p85. Receptor distal negative regulators are MAP4K1, which impairs the TCR pathway via phosphorylation of SLP76, and DGKα/ζ. The latter eliminates the PKC and RasGRP1 activating ligand DAG via phosphorylation. T cell activating cytokine signaling via the JAK/STAT pathway is negatively regulated by the phosphatase PTPN2 which dephosphorylates both JAK family proteins and STAT5. This figure was created with BioRender.com.

Figure 2

PGE2-mediated immunosuppressive, pro-tumorigenic signaling via EP2/EP4 receptors. PGE2 is released within the TME by various immune cells and cancer cells. By binding to the GPCRs EP2 and EP4, PGE2 mediates the induction of the coupled G-protein subunits G_sα and β/γ. This allows the activation of the PI3K-AKT pathway and the induction of the adenylate cyclase with subsequent cAMP release. High levels of cAMP mediate PKA kinase activation, which then can inhibit GSK, acting as a negative regulator of β-catenin. Next to β-catenin, other transcription factors are induced, including CREB and STAT3, resulting in the suppression of anti-tumor immune response and promotion of cancer development and progression. This figure was created with BioRender.com.

Figure 3

Negative regulators within the cGAS/STING and adenosine signal network. The cGAS-STING cascade is able to sense cytosolic DNA fragments that can originate from tumor cells. The enzyme cGMP-AMP synthase (cGAS) produces the second messenger cGAMP upon DNA binding, which binds and activates STING located at the ER. STING then translocates to perinuclear regions, where it binds and activates the kinase TBK1. The TBK1-STING complex is subsequently able to bind the transcription factor IRF3, which is being phosphorylated by TBK1. IFN3 then dissociates from the complex and induces the expression of several target genes, including IFNβ. As negative regulators of the cGAS-STING pathway, on one side the exonuclease TREX1 is in place, degrading cytosolic DNA, thereby reducing the ligand of cGAS. On the other hand, the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) mediates the degradation of the STING ligand cGAMP to AMP. With AMP being the source of adenosine production, ENPP1 contributes to the immunosuppressive adenosine pathway. Adenosine is elevated in the TME. It is generated from AMP via ectonucleotidase CD73. Next to the AMP source from ENPP1, the main source of AMP for adenosine production originates from the ATP breakdown mediated by CD39. The immunosuppressive effect of eADO is mediated via GPCRs A2aR (expressed on all immune cells) and A2bR (expression restricted to myeloid cells). On T lymphocytes A2aR mediates a suppression of TCR signaling resulting in an overall negative regulation of T cell activation, proliferation and survival. On DCs mainly A2bR signaling alters the expression of various immunomodulating factors, resulting in immunosuppressive downstream signaling events. This figure was created with BioRender.com.