The classical PI3K pathway

The PI3K signaling pathway is one of the most frequently altered signaling pathways in human cancer,^52–55 which can be activated by various growth factors/ligands specific to different RTKs, including members of the EGFR family, and the insulin and insulin-like growth factor 1 (IGF-1) receptor, FGF, etc.⁵⁶ LSCC has high rates of alterations in the PI3K pathway, and alterations were found in 68% of LSCC samples.⁵⁷ Activation of PI3K signaling pathway mediated through molecular aberrations is instrumental in promoting tumor development as well as resistance to antitumor therapies.^55,58 PI3K belongs to a family of lipid kinases, which are classified into three different classes based on structural features and lipid substrate preferences.⁵⁹ Class I PI3K is frequently implicated in cancer. Class IA consists of the PIK3CA, PIK3CB, and PIK3CD genes, and encode for the catalytic subunit of p110α, p110β, and p110δ, respectively. Class IB includes PIK3CG coding for p110γ.⁵⁴ Whereas p110α and p110β show broad tissue distribution, p110δ and p110γ are highly enriched in all leukocyte subtypes.⁶⁰

A common mechanism of PI3K activation in cancers is through the presence of mutations in the PIK3CA gene.⁶¹ The reported incidence of PIK3CA alterations in LSCC varies between 8 and 20% and the two main mutation types are canonical PIK3CA mutations and PIK3CA amplification.^62–64 The canonical PIK3CA mutations affect two different domains of p110α, the kinase domain and the helical domain.⁶⁵ These two types of mutations can activate the downstream signaling through two distinctive mechanisms. The kinase domain mutations can change the dynamics of the membrane-binding surface and affect the PIP₂ substrate.⁶⁶ The helical domain mutations (e.g., E542K, E545K) abrogate the inhibitory interactions between p110α and the N-terminal SH2 domain of the p85 regulatory subunit, leading to constitutive activity that mimics pTyr stimulation.^66,67

Mutations in other genes may also lead to abnormal activation of the PI3K pathway. Phosphatase and tensin homolog (PTEN) is a 9-exon tumor-suppressor gene located on chromosome 10q23. This gene encodes for a 403-amino acid protein with dual lipid and protein phosphatase utility which contains four functional domains: an N-terminal PI(4,5)P2-binding/phosphatase, domain, a C2 domain, a carboxyl-terminal tail domain (C-tail), and a PDZ-binding domain (PDZ-BD).⁶⁸ This protein classically dampens the PI3K/AKT/mTOR growth-promoting signaling cascade by directly dephosphorylating phosphatidylinositol-3,4,5-trisphosphate (PIP₃) and converting it back to the phosphatidylinositol-4,5-bisphosphate (PIP₂) inactive state.^69,70 Accordingly, PTEN dysfunction causes dysregulation of this and other pathways, resulting in tumorigenesis and cancer progression.^71–73 PTEN is mutated in 7–10% of LSCC^62,63,74 and these somatic mutations tend to be distributed across its 9 exons. Some tumor-associated missense mutations may lead to complete loss or severe impairment of the phosphatase activity of the encoded enzyme.^75–77 Many tumor-derived PTEN mutants retain partial or complete catalytic function, suggesting that alternative mechanisms can lead to the inactivation of PTEN.⁷⁸ In addition to its own genetic alterations, PTEN gene expression is also regulated at epigenetic,⁷⁹ transcriptional,^80–82 post-transcriptional^83–86, and post-translational^87–92 levels as well as by protein–protein interactions.^93,94

Partial loss of PTEN function can have dramatic effects on tumorigenesis and cancer progression,^71,72 reflecting the fact that PTEN is a haploinsufficient tumor suppressor. PTEN function is not often completely lost in cancer, providing an opportunity to reactivate its function as a mode of cancer treatment. It has been reported that a tumor-suppressive metabolic state is induced in transgenic mouse lines with the systemic elevation of PTEN.⁹⁵ In 2019, Lee et al.⁹⁶ reported a way to reactivate PTEN by inhibiting the MYC-WWP1 inhibitory pathway. In the study, they identified the HECT-type E3 ubiquitin ligase WWP1 as a physical PTEN interactor, the amplification, and overexpression of which may lead to pleiotropic inactivation of PTEN. A natural and potent WWP1 inhibitor, indole-3-carbinol (I3C), was also found to effectively suppress tumorigenesis driven by the PI3K-AKT pathway. Therefore, both genetic and pharmacological targeting of the MYC-WWP1 axis may be a viable approach for cancer patients driven by impaired PTEN function.

Although extensive research has been conducted on the PI3K pathway component genes as potential molecular therapeutic targets in human cancers in the past two decades, clinical success to date has been limited to the approval of the PI3K inhibitors for hematological malignancies and breast cancer.^34,97–101 Even among the four current FDA-approved PI3K inhibitors for the treatment of hematological malignancies, some of the indications have been withdrawn from the marketing authorization application.^102–105 Although some of the decisions were made, according to the manufacturing companies, based on business needs, this will certainly have implications for the future of PI3K inhibitors. In NSCLC, the early-phase clinical trials of PI3K inhibitors and dual PI3K/mTOR inhibitors have yielded negative results.^16–18 The modest therapeutic efficacy of PI3K inhibitors may be attributed to various reasons, including insufficient target inhibition, intrinsic and acquired drug resistance, and tolerability.¹⁰⁶ Unlike other oncogenes, such as EGFR in LUAD, the correlation between specific PI3K pathway mutations and drug sensitivity is not absolute.¹⁰⁷ This makes patient selection more complicated based on PI3K pathway mutation status. Another issue that cannot be overlooked about PI3K inhibitors is their on-target, off-tumor toxicity, particularly hyperglycemia and hyperinsulinemia which are observed as major dose-limiting toxicities.^98,108,109 Despite the benefits of PFS shown in several randomized clinical trials, the increased toxicities of PI3K inhibitors have raised concerns about the potential detriments of OS in the PI3K inhibitor arm.^{34,105,110–113} In blood cancers, future approvals of PI3K inhibitors by FDA should be supported by randomized data.¹¹⁴

It has been suggested that alternative dosing regimens which offer intermittent pathway inhibition can increase the therapeutic window without compromising therapeutic efficacy.^115–117 Another solution is to develop selective compounds that are more selective for mutant PI3K than wild-type PI3K. In the future, more durable therapeutic responses could be achieved by a more tailored PI3K-based therapies with a better understanding of the role of PI3K in cancer and surrounding environments.

Cell cycle in LSCC: the CDK4/6 pathway

The CDKN2A locus, located on human chromosome 9p21, is one of the most common genetic losses in human cancer.^118,119 TCGA profiling of 178 LSCC samples revealed that CDKN2A is inactivated in 72% cases of LSCC.⁴ The CDKN2A locus encodes two alternatively spliced proteins, p16INK4a (p16) and p14ARF (p14), which function as cell-cycle inhibitors. These two tumor-suppressor proteins function in distinct anticancer pathways: p16 regulates retinoblastoma (RB), and p14 regulates p53. RB is a tumor-suppressor protein which controls cell cycle by preventing entry into the DNA synthesis (S) phase of the cell-division cycle.¹²⁰ The p16 protein directly inhibits the activities of the cyclin D-dependent kinases, cyclin-dependent kinase (CDK) 4 and CDK6, thus maintaining RB in its dephosphorylated, anti-proliferative state, and leading to cell growth arrest.¹²¹ The tumor-suppressor protein p53 plays a pivotal role in regulating cell growth following exposure of cells to various stress stimuli.¹²² The p14 protein associates directly with murine double minute 2 (MDM2), a negative regulator of p53, preventing the export and degradation of p53.^123–126

At present, the therapeutic focus has been on leveraging CDK4/6 inhibition to activate RB and limit tumor cell proliferation to delay disease progression.^127,128 Interesting to note, the pan-caner analysis of the CDK4/6 pathway showed that CDKN2A loss and RB1 loss were mutually exclusive in most cancers that lose these genes at a significant level (>5%).¹²⁹ The proteogenomic portrait of LSCC revealed that loss of one of these two key CDK4/6 pathway inhibitors is a universal feature of LSCC.³⁸ However, CDK4/6 inhibitors have shown minimal efficacy in LSCC clinical trials.^19,130–132 Phospho-RB levels have been shown to be correlated with response to CDK4/6 inhibitors in various LSCC cell lines.³⁸ The heterogeneity of RB expression and phosphorylation may provide a reasonable explanation for the diverse responses toward CDK4/6 inhibitors. The screening of tumors based on the downstream functional assessment (i.e., RB expression and phosphorylation) may identify tumors that are sensitive to CDK4/6 inhibitors.

VEGF-VEGFR signaling in LSCC

Vascular endothelial growth factor (VEGF, here referred to as VEGF-A) is a member of a protein family that also includes VEGF-B, VEGF-C, VEGF-D, VEGF-E (a virally encoded protein), and placental growth factor (PIGF, also known as PGF).¹³³ VEGF-B has multifaceted and context-dependent functions that safeguard the balance between blood vessel growth and degeneration to ensure normal blood vessel density and integrity.¹³⁴ VEGF-C and VEGF-D are mainly implicated in lymphangiogenesis.¹³⁵ As VEGF-A plays a dominant role in regulating angiogenesis and disease, it is referred to as VEGF in this review. Alternative exon splicing causes multiple isoforms of VEGF which are characterized by their differential ability to bind heparin.¹³⁶ VEGF binds to both VEGF receptor 1 (R1) and VEGFR2 while VEGFR2 is the main receptor for VEGF.^137,138 VEGF isoforms can also interact with the neuropilin co-receptors (NRP1 and NRP2).^139,140 During tumorigenesis, angiogenesis plays a key role in maintaining the expansion in tumor. Most human tumors overexpressed VEGF mRNA, and its expression correlates with invasiveness, increased vascular density, metastasis, tumor recurrence and poor prognosis.¹⁴¹ Accordingly, several strategies that target this VEGF-VEGFR signaling has been devised.^142,143

Neutralizing monoclonal antibodies (mAbs) against VEGF have shown great effect in preclinical studies¹⁴⁴ and were the first type of antiangiogenic drugs that entered the market. In 2004, bevacizumab was approved by the FDA for the treatment of metastatic colorectal cancer based on the results of AVF2107 clinical trial.¹⁴⁵ However, as the benefits of bevacizumab extended to other malignancies, including non-squamous NSCLC, renal cell carcinoma, ovarian cancer, and cervical cancer,¹⁴² LSCC is not one of them, as clinical trials have shown that bevacizumab increases the risk of life-threatening pulmonary hemorrhages in squamous cell carcinomas.^146,147 Another antiangiogenic agent, ramucirumab, a human IgG1 monoclonal antibody targeting the extracellular domain of VEGFR2, is currently the only antiangiogenic agent that is approved by FDA for the treatment of LSCC. Based on the results of phase III REVEL clinical trial,¹⁴⁷ ramucirumab plus docetaxel is recommended as a subsequent therapy option for metastatic NSCLC following disease progression on or after platinum-based chemotherapy.¹⁴⁸

FGFR1 pathway

Fibroblast growth factor receptor 1 (FGFR1) belongs to the FGFR family of receptor tyrosine kinases (RTKs), which consists of four members: FGFR1 to FGFR4. All these four members share a canonical RTK architecture, consisting of a large ligand-binding extracellular domain that comprises three immunoglobulin-like domains (D1-3) followed by a single transmembrane helix and an intracellular domain containing the catalytically active “split” tyrosine-kinase domain.^149,150 There is also a fifth related receptor, FGFR5 (also known as FGFRL1), which lacks the cytoplasmic tyrosine-kinase domain.¹⁵¹ The native ligand of FGFRs is fibroblast growth factors (FGFs), which can be divided into two categories: hormone-like FGFs (i.e., FGF19, 21, and 23) and canonical FGFs (i.e., FGF1-10, 16–18, and 20).^150,152 The intracellular signaling of the FGFR pathway is primarily mediated mainly through four key downstream pathways: RAS-RAF-MAPK pathway, PI3K-AKT, signal transducer and activator of transcription (STAT), and phospholipase Cγ (PLCγ)^153–155 (Fig. ​(Fig.1).1). Dysregulation of FGFR signaling promotes the proliferation,¹⁵⁶ survival¹⁵⁷ and development of drug resistance¹⁵⁸ in tumor cells, as well as the development of angiogenesis¹⁵⁹ and immune evasion in the tumor microenvironment (TME).¹⁶⁰ These findings make FGFR pursued as a potential therapeutic target and support the development of FGFR-targeting anticancer agents.

FGFR1 amplifications are the predominant type of FGFR mutation, occurring in nearly 20% of LSCC patients.^160,161 Although the studies in preclinical models have suggested that FGFR inhibitors may be a viable therapeutic option in this cohort of patients,^161,162 a number of FGFR-specific small molecular inhibitors tested in phase I and phase II trials have shown modest effects with overall response rates of 8–15%.^11–15 The results from these trials suggest that FGFR1 amplification is not a reliable predictor of response to FGFR1 inhibitors and that FGFR1 mutations have a more complex impact in NSCLC than EGFR-mutated or ALK-rearranged NSCLC.¹⁶³ A previous study has found that elevated FGFR1 mRNA and/or protein expression was often independent of FGFR1 amplification.¹⁶⁴ Future studies are needed to clarify the role of FGFR1 signaling in the pathogenesis of LSCC.

EGFR pathway

EGFR belongs to the HER/erbB family of RTKs, which includes HER1 (EGFR/erbB1), HER2 (neu, erbB2), HER3 (erbB3), and HER4 (erbB4). All members display similar structures: an extracellular, cysteine-rich ligand-binding region, a single alpha-helix membrane-spanning region and a cytoplasmic tyrosine-kinase-containing domain.¹⁶⁵ The intracellular signaling of EGFR pathway is mediated mainly through the RAS/MAPK pathway, the PI3K pathway, and the STAT pathway.^166,167 Downstream EGFR signaling ultimately leads to increased proliferation,¹⁶⁸ angiogenesis,¹⁶⁹ metastasis,¹⁷⁰ and decreased apoptosis.¹⁷¹ Alterations in EGFR signaling pathways result in constitutive activation of its kinase activity and the inhibition of tumor apoptosis, leading to a poor clinical outcome.^172,173 All these findings make EGFR pursued as therapeutic targets and support the development of EGFR-targeting anticancer agents.¹⁷⁴

The reported rate of EGFR mutation in LSCC patients is 4.2–7%,^4,175,176 which is much lower compared with LUAD patients. In previous prospective phase III clinical trials assessing the efficacy of first-line EGFR-TKIs in the treatment of NSCLC, only 27 cases of LSCC patients with EGFR mutation were identified in six clinical trials, which were further randomized into two groups.^177–182 This limited number of LSCC cases makes it hard to assess the benefits of EGFR-TKIs for EGFR-mutated LSCC in prospective studies. Subgroup analysis in the BR.21 and SATURN clinical trials showed that erlotinib was effective in unselected LSCC patients.^183,184 A meta-analysis also confirmed that EGFR-TKIs demonstrated an improved OS and PFS compared to placebo in unselected patients with advanced LSCC.¹⁸⁵ Based on previous retrospective matched-pair studies,^186,187 EGFR-TKIs were less effective in EGFR-mutant LSCC than in LUAD but still had clinical benefits for LSCC patients. Another retrospective study found that in Chinese female EGFR-mutant LSCC, EGFR-TKIs conferred longer PFS and OS than chemotherapy, but the survival was similar with patients without EGFR mutations.¹⁸⁸ In conclusion, for EGFR-mutant LSCC, EGFR-TKIs can improve the outcomes of these patients compared with chemotherapy, but its efficacy is not as robust as that of EGFR-TKIs for EGFR-mutant LUAD.

Notably, EGFR protein was significantly upregulated in the squamous cancers but not in LUAD,³⁸ although many activating EGFR mutations occurred in LUAD. This EGFR amplified LSCC cohort did not show elevated EGFR pathway activity,¹⁸⁹ but displayed a high correlation with mRNA abundance of the five EGFR ligands. This is consistent with the results in HNSCC,¹⁹⁰ which indicates a squamous cell carcinoma feature that EGFR ligand abundance drives the activity of EGFR pathways. It suggests that EGFR ligand abundance, rather than EGFR amplification, might be a better predictor for EGFR inhibitor response in this population of LSCC patients.

SOX2

Given that SOX2 is amplified in various types of cancer and involved in tumorigenesis via complicated signaling pathways and protein–protein interactions, targeting SOX2 is a promising strategy for anticancer therapy.⁴⁰ Previously, as a transcription factor, SOX2 was deemed undruggable because of its absence of active sites or allosteric regulatory pockets to be targeted by small molecule inhibitors (SMIs).²⁴⁴ Therefore, studies targeting SOX2 in anticancer therapy has been focusing on the upstream and downstream signaling of SOX2. Recently, Liu et al.²⁴⁵ reported the development of a platform using the technique of proteolysis-targeting chimeras (PROTACs), which is able to selectively degrade the transcription factors of interest. This generalizable platform may help target SOX2 as an effective anticancer therapy.

Np63

The main isoform of p63 expressed in adult squamous tissues is Np63.²⁴⁶ For squamous cell carcinomas (SCCs), Np63 acts as a proto-oncogenic transcription factor and the master regulator of SCC formation.^247–250 The oncogenic potential of Np63 is related to its direct competition with p53, TAp63, and TAp73 on the same p53 responsive elements and the consequent inhibition.^251,252 High levels of endogenous ΔNp63 protein abundance are essential to induce and maintain SCC tumors.^247,253 Acute gene ablation of ΔNp63 in an autochthonous SCC model could induce rapid tumor regression.²⁵³ Besides, ΔNp63 is also found to regulate chemoresistance in SCCs by controlling the expression of DNA repair genes.^254,255 Collectively, these findings implicate that ΔNp63 is a promising therapeutic target in LSCC. As a transcription factor, ΔNp63 was considered undruggable, as with most transcription factors which lack suitable domains for the binding of SMIs.²⁴⁴ The development of a generalizable platform by Liu et al.²⁴⁵ based on the technique of PROTACs, which is able to selectively degrade the transcription factors of interest, may provide new strategies to target Np63. However, it is of note that Np63 is associated with the regulation of a massive subset of different genes and cellular processes, which makes complete blocking of Np63 almost impossible.

USP28

Ubiquitin-specific peptidase 28 (USP28) belongs to the largest deubiquitinating enzyme family, which removes ubiquitin from the ubiquitin conjugates.²⁵⁶ ΔNp63 is tightly regulated at the protein level by the ubiquitin-proteasome system, which can be targeted by multiple E3 ligases.²⁵⁷ USP28 is highly abundant in SCCs and correlates with poor prognosis.²⁵⁸ In SCCs, USP28 could stabilize ΔNp63 and maintain elevated ΔNp63 levels by counteracting its proteasome-mediated degradation.²⁵⁸ The researchers further confirmed that the pharmacologic inhibition of USP28 showed a selective anti-proliferative response of SCC cells.²⁵⁸ In addition to its tumor-suppressive function, inhibition of USP28 in ΔNp63 expressing SCC could sensitize SCC cells to cisplatin treatment by toning down the DNA damage response pathways.²⁵⁹ Taken together, these data show that USP28-ΔNp63 axis is required in the maintenance of SCC identity and control of SCC marker gene.

USP28 stabilizes ΔNp63 independently of FBXW7,²⁵⁸ which is a component of SCF (complex of SKP1, CUL1, and F-box protein)-type ubiquitin ligases.²⁶⁰ FBXW7 is a tumor suppressor that binds to key regulators of cell division and growth, including cyclin E, MYC, JUN, and Notch, most of which are proto-oncogenes that are closely related to the pathogenesis of human cancers.²⁶¹ Recurrent mutations in the FBXW7 tumor-suppressor gene have been reported in LSCC.^20,262 FBXW7 and USP28 are closely related in that USP28 could lead to FBXW7 substrate accumulation (either via destabilization of FBXW7 or via stabilization of both FBXW7 and its substrates).^263,264 Therefore, targeting USP28 to destabilize the substrates of FBXW7 represents a promising strategy to inhibit the function of MYC and other oncogenic regulators.

Inhibition of USP28 is particularly effective in mouse LSCC models, resulting in dramatic tumor regression.^258,265 The USP28 inhibitor used by Prieto-Garcia et al.²⁵⁸ was AZ1, a dual USP25/USP28 inhibitor, while the USP28 inhibitor FT206 used by Ruiz et al.,²⁶⁵ preferentially inhibits USP28 compared to USP25. Despite evidence that USP25 is an oncoprotein,²⁶⁶ its oncogenic function in LSCC is still enigmatic. There is currently no specific inhibitor of USP28 mainly due to the highly similar catalytic structure of USP25 and USP28. In the future, with the help of novel drug development technologies, USP28 inhibitors may become a promising therapeutic option for LSCC, but further clinical trials are still needed.

Survivin

Survivin (also known as BIRC5) has been a well-known cancer therapeutic target since its discovery over 20 years ago.²⁶⁷ Because of its essential role in cell mitosis and inhibition of apoptosis,^268–270 as well as its variable expression levels in cancer and normal cells,²⁷¹ survivin appears be a ideal candidate for anticancer therapy. However, no survivin-specifc drugs have yet reached the clinic. SMIs and inhibitory peptides targeting survivin for NSCLC have been explored in clinical trials but have shown modest or no improvement.^272–274

Recently, Satpathy et al.³⁸ identified ΔNp63-low LSCC which showed no elevation at RNA or protein levels. Accordingly, they also discovered a substantial number of LSCC cell lines with low ΔNp63 expression which were significantly more vulnerable to the survivin inhibitor YM-155. These findings may provide new strategies for selecting LSCC patients based on the TP63 status, which may have better response to survivin inhibition.

NSD3: the neighboring gene of FGFR1

A recent proteogenomic portrait of LSCC suggested that WHSC1L1 (NSD3), but not FGFR1, may be the critical driver oncogene within the recurrent focal amplicon (8p11.23).³⁸ NSD1, NSD2, NSD3, and ASH1L are four related enzymes in mammals which can synthesize the euchromatin-associated H3K36me2 modification.²⁸⁶ NSD3 dimethylates (adds two methyl groups to) the 36th amino acid residue in histone H3 (a lysine residue dubbed H3K36).²⁸⁷ This process, in which various chemical groups are covalently added to, or removed from, the DNA bases and the tails of the histones is referred to as epigenetic modifications.²⁸⁸ Amplification of NSD3 and its immediate neighbors (e.g., FGFR1), located on the chromosomal region 8p11-12, is one of the frequent molecular alterations in LSCC²⁸⁹ and has been implicated in the etiology of LSCC.^290,291 In contrast to FGFR1, gene amplification of NSD3 correlates strongly with increased mRNA expression.²⁹¹ Accordingly, a recent study has also shown that depletion of NSD3 in the 8p11-12 amplified LSCC cell lines and mouse model significantly attenuated tumor growth.⁴⁴ This study also confirmed the ability of NSD3 to cooperate with SOX2 to transform human tracheobronchial epithelial (AALE) cells which further verified that NSD3 could promote human LSCC tumorgenesis⁴⁰ (Fig. ​(Fig.4a4a).

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Fig. 4

Schematic diagram of the different roles for epigenetic therapeutic targets in LSCC. a A recent study suggested that NSD3, the neighboring gene of FGFR1, rather than FGFR1, was the critical driver oncogene within this recurrent focal amplicon of 8p11-12 genomic region. The amplification of NSD3 leads to increased NSD3 expression, thus increasing the synthesis of H3K36me2. Less common than the amplification of 8p11-12 and NSD3 expression, the GOF variant NSD3 was also present in LSCC. These two works together to increase H3K36me2, stimulating transcription of oncogenic targets, including mTOR pathways and MYC-associated pathways. This process rendered the tumor NSD3-addicted, which could be inhibited by BETi. b SOX2 and BCL11A are both identified as LSCC oncogenes. The BCL11A-SOX2 transcriptional program is crucial for the maintenance of a squamous phenotype. SETD8 is a monomethyltransferase, whose gene is regulated by SOX2 and BCL11A. The inhibition of SETD8 selectively limits LSCC tumor growth. c LSD1 could promote tumorigenesis in two different ways. The first way is demethylase-dependent. In SOX2-expressing tumor cells, LSD1 inhibition will induce increased H3K9me1/me2. The repressive H3K9 methylations act on the SOX2 gene, leading to SOX2 downregulation, reduced oncogenic potential, and increased cellular differentiation. The second way is demethylase-independent. In cells with a low level of LSD1, FBXW7 forms a dimer, which promotes ubiquitylation for proteasomal degradation of oncoprotein substrates, thus suppressing cell outgrowth. In cancer cells with overexpressed LSD1, the FBXW7 dimerization is blocked by LSD1 binding to FBXW7 in a demethylase-independent manner. FBXW7 self-ubiquitylation will then be triggered, followed by degradation by proteasome as well as lysosome in a p62-dependent pathway. d EZH2 is an enzymatic subunit of PRC2, which also includes EED, SUZ12, and RBBP4/7. The SET domain of EZH2 is responsible for the catalyzes the mono-, di-, and trimethylation of H3K27 from the universal methyl donor SAM, after which SAM becomes SAH. EZH2 also has noncanonical functions with its hidden TAD. The EZH2 TAD directly interacts with cMyc and other activators, including p300 and SWI/SNF. GOF gain-of-function, PRC2 polycomb repressive complex 2, SAH S-adenosyl-l-homocysteine, SAM S-adenosyl-l-methionine, TAD transactivation domain

Given that currently there is no catalytic inhibitors of NSD3 available in physiological settings, the researchers in that study also found that the four bromodomain inhibitors (BETi) exhibited the highest differential lethality over cells with mutated NSD3.⁴⁴ This clinical actionable vulnerability, accompanied with findings of Li et al.²⁹² who used cryo-electron microscopy to solve the structures of normal and oncogenic mutant forms of NSD3 bound to a nucleosome, will certainly provide valuable information for the design and development of drugs for treating LSCC as well as other NSD-driven diseases.

SETD8

SETD8 (also known as PR-Set7, SET8, and KMT5A) is currently the only known H4K20me1 monomethyltransferase, which is implicated in the regulation of multiple biological activities, including DNA replication, DNA damage repair, cell-cycle progression, and transcription regulation.^293,294 During mitosis, SETD8 is concentrated in the nucleus during G1 and G2 phases and is degraded through ubiquitination at G1/S transition.²⁹⁵ Besides H4K20, SETD8 can also regulate the tumor-suppressor protein p53 and proliferating cell nuclear antigen (PCNA), which are closely related to carcinogenesis.^296–298 SETD8 is implicated in cancer proliferation, migration, invasiveness, and oncogenesis, associated with a poor outcome.^299,300

In the study by Lazarus et al.,⁴⁵ BCL11A, which encodes a transcriptional regulator, was identified and characterized as a LSCC oncogene. Along with SOX2, which was also regarded as an oncogene in LSCC,⁴⁰ this BCL11A-SOX2 transcriptional program provides a potential therapeutic window for LSCC. To disrupt this BCL11A-SOX2 transcriptional program, a Gene Ontology (GO) was performed and the SETD8 gene is selected, which is regulated by both BCL11A and SOX2.⁴⁵ Knockdown of SETD gene could selectively inhibit LSCC tumor growth, but not LUAD cell. Besides, SETD8 inhibition also sensitizes LSCC cell lines to chemotherapy. Collectively, this study highlights the BCL11A-SOX2 transcriptional program as a novel target for LSCC and suggests the monomethyltransferase SETD8 as a potential downstream target⁴⁵ (Fig. ​(Fig.4b4b).

LSD1

Lysine-specific demethylase 1 (LSD1, also known as KDM1A, KIAA0601, BHC110, and AOF2) is one of the SOX2-related targets that has been extensively studied. LSD1 is the first identified histone demethylase, which has the dual substrate specificity to catalyze the demethylation of histone 3 lysine 4 (H3K4me1/2) and H3K9me1/2 for transcriptional repression.^301–303 The expression of LSD1 histone demethylase was reported to be significantly elevated in SOX2-expressing LSCC.⁴⁶ LSCC cell lines with amplified SOX2 gene are particularly sensitive to LSD1 inactivation, whereas SOX2-negative cells are not. The regulation of SOX2 gene by LSD1 is directly through the bivalent H3K4 and H3K9 methylations. As a key regulator of SOX2, which is a lineage-survival oncogene of LSCC,⁴⁰ LSD1 can serve as a specific and selective target for the treatment of LSCC.

In addition to its demethylase activity, the demethylase-independent activity of LSD1 has also been implicated in carcinogenesis.^47,304,305 LSD1 can act as a pseudosubstrate of FBXW7. FBXW7 is a typical tumor suppressor that targets many oncoproteins for ubiquitylation and degradation.²⁶¹ FBXW7 dimerization is disrupted by the binding of FBXW7 and LSD1 which promotes FBXW7 self-ubiquitylation and degradation through proteasome and lysosomal pathways, independent of the demethylase activity of LSD1, thus leading to accelerated growth⁴⁷ (Fig. ​(Fig.4c).4c). The discovery of this demethylase-independent activity of LSD1 implicates that the efforts to develop LSD1 inhibitors should be extended to directly target LSD1 rather than just inhibit its demethylase activity, which should harbor broader utility in anticancer therapy.

Currently, many LSD1 inhibitors are tested in phase I/II clinical trials,³⁰⁶ although most inhibitors were based on blocking its demethylase activity. However, the ineffectiveness of catalytic inhibition of LSD1 has been noticed in certain cancers.^307,308 Therefore, targeting LSD1-involved protein interactions with the emerging technologies of PROTACs,³⁰⁹ not confined to the inhibition of its demethylate activity, may be a novel anticancer therapy in cancers with LSD1 overexpression like LSCC.

ICB therapy

Immune-checkpoint blockade is one of the most promising approaches to activating antitumor immunity. The immune-checkpoint pathways are involved in the major mechanisms underlying tumor immune evasion. Physiologically, these immunosuppressive signaling pathways play important roles in maintaining self-tolerance to prevent autoimmunity, limit immune-mediated tissue damage, and control the resolution of inflammation.^332,333 Cancer cells may take advantage of these immune checkpoints to disguise themselves from body immune system.^334,335 Among these immune checkpoints, cytotoxic T-lymphocyte antigen 4 (CTLA-4) and PD-1/PD-L1 axis are the most potent examples of T-cell immune-checkpoint molecules. The ICB therapies which were approved by FDA or National Medical Products Administration (NMPA) for LSCC are summarized in Table ​Table11.

CTLA-4: the first clinically targeted immune-checkpoint receptor

CTLA-4 is a homolog of CD28 and binds both B7-1 (also known as CD80) and B7-2 (also known as CD86) with much higher affinity than CD28.^336–339 The CTLA-4 and CD28 genes are located in the same region of chromosome 2 (2q33.2) and are expressed by both CD4⁺ and CD8⁺ T cells with opposing functions in T-cell activation.^337,339 Through interacting with a pair of ligands (B7-1 and B7-2) expressed on antigen-presenting cells (APCs), including macrophages, dendritic cells (DCs) and B cells, CD28 mediates T-cell activation by co-stimulating T-cell receptor (TCR) signaling while the interaction of the ligands with CTLA-4 serves to inhibit T-cell response.³⁴⁰ These regulatory effects of CTLA-4 mainly restrict the expansion of CD4⁺ helper T cells while boosting regulatory T cells (Tregs),^334,341 thus leading to a pro-tumor immunosuppressive phenotype.³⁴²

The recognition of CTLA-4 as a negative regulator of T-cell activation makes antagonizing CTLA-4 a reasonable method to enhance the antitumor immunity of T cells.³⁴³ Initial preclinical studies found that CTLA-4 blockade enhanced antitumor immunity and caused regression of immunogenic tumors without inducing substantial autoimmunity.^344,345 Based on these preclinical findings, several clinical trials have been conducted to evaluate the therapeutic efficacy of CTLA-4 antibodies in tumors,^346–349 which finally led to the FDA approval of ipilimumab by FDA for the treatment of advanced melanoma. However, the impressive effects of ipilimumab in melanoma patients did not proceed in renal cell carcinoma,³⁵⁰ NSCLC,³⁵¹ small-cell lung cancer³⁵² and prostate cancer.³⁵³ Another CTLA-4-blocking antibody, tremelimumab, has not received FDA approval since it did not improve survival compared to chemotherapy in metastatic melanoma.³⁵⁴ As the first immune-checkpoint inhibitor, ipilimumab is also currently the only CTLA-4-blocking antibody that has gained approval for anticancer treatment. No CTLA-4 inhibitors have been approved as monotherapy or in combination therapy with chemotherapy for the treatment of NSCLC (Table ​(Table11).

The fact that anti-CTLA-4 antibodies are capable to induce long-term immunity in cancer patients demonstrates that CTLA-4 remains an important immunotherapy target.^355,356 Nevertheless, CTLA-4-targeting inhibitors have not reached its full potential, as evidenced by high rates of immunotherapy-related adverse effects (irAEs) and relatively low response rates. The strong irAEs of ipilimumab limit the doses tolerated by cancer patients. Both anti-PD-1/PD-L1 antibodies and anti-CTLA-4 antibodies have irAEs, while the effects of anti-CTLA-4 therapy are generally more severe.^357–360 The dose-limiting toxicity of ipilimumab presented an opportunity of developing the next-generation molecules with wider therapeutic window.^361–363 Recently, additional mechanisms were raised to explain the immunotherapeutic effects of anti-CTLA-4 mAbs, including depletion of regulatory T cells (Tregs) in TME.^{341,364–367} According to Du et al.,³⁶⁸ ipilimumab remains full activity without blocking B7-CTLA-4 interaction. In their studies, the humanized antibodies they developed without blockade of the B7-CTLA-4 interaction were as effective as ipilimumab at causing rejection of cancer. To further confirm that this tumor rejection was induced by Tregs depletion through antibody-dependent cellular cytotoxicity (ADCC), concurrent administration of anti-FcR antibodies treatment completely abolished the anticancer effect of ipilimumab. Collectively, these findings suggest that the selective Treg depletion in the tumors may be the primary mechanism of antitumor effect of anti-CTLA-4 antibody rather than the blockade of B7-CTLA-4 interactions³⁶⁹ (Fig. ​(Fig.55).

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Fig. 5

Roles of Fcγ receptors in anti-CTLA-4 function. Selective deletion of Tregs in the tumor microenvironment results in tumor immunity (left). Expressing higher levels of CTLA-4 than effector T cells, intratumoral Tregs are selectively depleted through ADCP by macrophages and/or ADCC by NK cells. In T-effector cells, T-cell activity is enhanced by the recognition of MHC-Ag by the TCR in the presence of an anti-CTLA-4 antibody that had co-engaged with FcγR on APCs (right). ADCC antibody-dependent cellular cytotoxicity, ADCP antibody-dependent cellular phagocytosis, APC antigen-presenting cells, MHC-Ag major histocompatibility complex-antigen peptide complexes major histocompatibility complex-antigen peptide complexes, NK cells natural killer cells, TCR T-cell receptor

Many new types of anti-CTLA-4 antibodies have been developed to increase antitumor effect, reduce side effects, or both. Increasing the ability of Fc to bind to FcR is one of the strategies to enhance the antitumor effect which can be achieved through a non-fucosylated derivative of ipilimumab (BMS986218) or an engineered Fc variant of an anti-CTLA-4 antibody (AGEN-1181or its mouse surrogate).³⁷⁰ The next-gen anti-CTLA-4 mAb, ONC-392, which effectively and selectively eliminates Tregs, has been granted Fast Track designation granted by FDA for monotherapy in PD-(L)1-resistant NSCLC.³⁷¹ Different from other anti-CTLA-4 mAbs being tested, the pH-sensitivity nature of ONC-392 avoids antibody-triggered lysosomal degradation of CTLA-4, thereby reducing toxicity and exerting its anticancer potential.³⁶³ ONC-392 is currently being evaluated in Phase I clinical trial (PRESERVE-001; NCT04140526) for advanced solid tumors and NSCLC. An additional approach to moderate the adverse event profile of anti-CTLA-4 is to limit the CTLA-4 blockade within the tumor. For example, a “proform” of ipilimumab (BMS-986249) was synthesized, which was designed to remain inert in the periphery, but have activity restored when unmasked by tumor-associated proteases.³⁷⁰ Another approach is to generate a pH-selective form of ipilimumab, which could preferentially and reversibly target the acidic TME over the neutral periphery.³⁶²

PD-1 axis

PD-1 axis was the second immune-checkpoint pathway targeted for ICB therapy. In 2014, fully humanized anti-PD-1 mAbs pembrolizumab and nivolumab became the first PD-1 targeted therapeutics approved by FDA for refractory and advanced melanoma.^{357,372–374} Although anti-PD-(L)1 therapy entered the market later than anti-CTLA-4 therapy, PD (L)-1 blockade have shown broader clinical utility than anti-CTLA-4 treatment. For LSCC, a number of anti-PD-(L)1 therapeutics have been approved by FDA and NMPA as monotherapy or in combination therapy with chemotherapy (Table ​(Table11).

PD-1 was first identified as a putative mediator of apoptosis in 1992,³⁷⁵ and its role in maintaining peripheral tolerance by serving as a negative regulator of immune responses was elucidated in 1999 when Nishimura et al.³⁷⁶ found that PD-1-deficient mice developed a late onset of lupus-like autoimmune disease. Nearly at the same time, Dong et al.³⁷⁷ revealed a new member of the B7 family which might be involved in the negative regulation of cell-mediated immune responses. In the next year, this new member of the B7 family was confirmed to be the ligand of PD-1 (PD-L1) and an inhibitor of T-cell activation.³³⁵ PD-L2, a second ligand with higher affinity for PD-1, was also identified.^378,379 Subsequent work found out that PD-L2 could have both co-stimulatory and co-inhibitory functions depending on the receptor and context.³⁸⁰ After being implicated in the negative regulation of T cells, the PD-1 axis was regarded as an active target of developing anticancer therapies. Multiple preclinical studies have showed that the PD-1 axis in the tumor causes the resistance to immune-mediated cytolysis, while blocking PD-L1 or PD-1 with specific mAbs in tumors could reverse tumors’ inherent resistance to cytotoxicity by T cells.^381–384 However, solely blocking PD-L2 did not demonstrate any antitumor effect.³⁸⁵ Following the success of preclinical studies, mAbs targeting the PD-1 axis were designed and showed remarkable efficacy in clinical trials. In a head-to-head comparison for PD-L1 expressing advanced NSCLC, monotherapy with the PD-1 inhibitor pembrolizumab showed significantly better OS and lower incidence of adverse events than chemotherapy.^386,387 The PD-L1 inhibitor atezolizumab also resulted in significantly longer OS than platinum-based chemotherapy in NSCLC patients with high PD-L1 expression.^27,388

As studies in immunotherapy increase, difference between the clinical effect of anti-PD-1 and anti-PD-L1 has been reported. Such disparities have drawn the attention of clinicians and a better understanding of this discrepancy may guide us for a better administration of these drugs. Currently there are no head-to-head comparisons of anti-PD-1 mAbs and anti-PD-L1 mAbs in clinical trials. In a systematic review and meta-analysis, Duan et al.³⁸⁹ adjusted indirect comparisons based on a well-designed mirror principle to minimize the potential bias and found out that anti-PD-1 mAbs appeared to exhibit significantly greater OS compared with anti-PD-L1 with a comparable safety profile in patients with solid tumors. The possible reason for the improved efficacy of anti-PD-1 mAbs compared with anti-PD-L1 mAbs may come from the mechanisms of PD-1 and PD-L1 blockade in anticancer therapy. Anti-PD-1 mAbs can bind to PD-1 and further block the interaction between PD-1 and its ligands (PD-L1 and PD-L2), while the PD-1/PD-L2 axis remains intact and exerts its immune suppressive functions when PD-L1 is blocked by anti-PD-L1 mAbs. Nevertheless, the blockade of PD-1 may shift the balance of the binding of PD-L2 with its other partner, repulsive guidance molecule b (RGMb), which can lead to pneumonitis.³⁸⁰ This is also confirmed by the fact that patients treated with PD-1 inhibitors have a higher incidence of pneumonitis than patients who received PD-L1 inhibitors.^390,391

Although great success has been achieved in the treatment of LSCC with the advent of PD-1 axis inhibitors, the ORR of PD-1 axis inhibitor in the treatment of advanced NSCLC is ~30%.^27,387 Therefore, it is of utmost importance for the establishment of effective biomarkers for predicting the efficacy of anti-PD-1 axis agents. The assessment of PD-L1 expression on tumor cells is a logical biomarker for the prediction of treatment response to anti-PD-1 or anti-PD-L1 therapies. A real-world study in China has found out that LSCC patients were associated with higher incidence rate of positive PD-L1 expression, suggesting a benefit of using ICIs in LSCC patients.³⁹² Although PD-L1 immunohistochemistry (IHC) plays an important role in patient stratification in clinical trials of anti-PD-1 or anti-PD-L1 therapies, it has poor reliability as a biomarker for anti-PD-1 or anti-PD-L1 therapies, as patients with negative PD-L1 expression can still benefit from anti-PD-1 or anti-PD-L1 therapies.^393–395 Beyond PD-L1 expression, several other biomarkers have also successively predicted the efficacy of ICB therapy to certain extent. Among them, tumor mutational burden (TMB), gene expression profiling (GEP), and multiplex immunohistochemistry/immunofluorescence (mIHC/IF) are mostly used.³⁹⁶ Due to the lack of accurate assessment of response, future improvements in diagnostic accuracy may be achieved through a multiple incorporation of existing markers and newly discovered markers.^396–400

LAG3

Lymphocyte activating gene 3 (LAG3, also known as CD223), first discovered in 1990,⁴⁰¹ is a transmembrane molecule that is expressed on CD4⁺ and CD8⁺ T cells, natural killer T (NKT) cells, natural killer (NK) cells, plasmacytoid dendritic cells (pDCs) and Tregs.^402,403 The LAG3 gene is located on human chromosome 12 (12p13.31), adjacent to the coding region of CD4.⁴⁰⁴ The LAG3 protein and CD4 protein share approximately 20% similarities in their amino acid sequences, which is mostly pronounced on their extracellular regions.^404,405 Due to this similarity in extracellular structures, like CD4, LAG3 can also bind to major histocompatibility complex class II (MHC-II) proteins, but with higher affinity, which is also the canonical ligand for LAG3.⁴⁰⁶ Once LAG3 binds to MHC-II proteins, the inhibitory signals are transmitted through its cytoplasmic domain, thereby downregulating T-cell function.⁴⁰⁷ Several other ligands were also found to interact with LAG3, including Galectin-3⁴⁰⁸ (Gal-3), liver sinusoidal endothelial cell lectin^409,410 (LSECtin), and fibrinogen-like protein 1⁴¹¹ (FGL1).

The fact that LAG3-deficient T cells show enhanced homeostatic expansion suggests the inhibitory role of LAG3 in immune responses.^412,413 LAG3 is co-expressed with other inhibitory receptors, such as PD-1, on CD8⁺ tumor antigen-specific T cells under chronic tumor antigen stimulation, which leads to T-cell exhaustion.^403,414 LAG3 expression was also confirmed to play an important role in supporting Tregs activity.⁴⁰² In intratumoral Tregs, LAG3 is expressed at a higher level than in Tregs found in peripheral or normal tissue.^415,416 Multiple LAG3-modulating candidates have been developed, including LAG3-inhibiting antibody and LAG3 fusion protein.⁴¹⁷ However, LAG3 monotherapy in several mouse models has shown limited antitumor effect with slightly reduced tumor growth, whereas LAG3/PD-1 co-blockade has shown much stronger synergistic antitumor effects.^418–422 Several anti-LAG3 antibodies are currently being evaluated in clinical trials, in which relatlimab is the furthest along in clinical development among all the anti-LAG3 mAb. On March 18, 2022, the combination therapy of relatlimab and nivolumab was approved by FDA for the treatment of unresectable or metastatic melanoma, making LAG3 the third FDA-approved immune checkpoint that was approved by FDA after CTLA-4 and PD-1 axis.^423,424 This approval of LAG3 mAb marks an exciting beginning for this inhibitory receptor but many aspects of its biological functions still remain enigmatic. New ligands of LAG3 are still emerging and the effects of LAG3 on immune cells remain to be fully characterized.⁴¹⁷ For LSCC, several anti-LAG3 antibodies and bispecific antibodies are being evaluated in phase I and phase II clinical trials (Table ​(Table22).

ICB and PI3K pathway inhibition

The hyperactive PI3K signaling, whether it is the consequence of PI3KCA mutations or PTEN deletions, can promote the establishment of tumor suppression by developing tumors,⁴⁶⁷ suggesting the potential use of PI3K inhibitor to enhance the efficacy of immunotherapy in the clinic (Fig. ​(Fig.6a).6a). In preclinical models of melanoma, loss of PTEN in tumor cells inhibits T-cell-mediated tumor killing and restricts T-cell trafficking into tumors.⁴⁶⁸ A number of immunosuppressive cytokines, including CCL12 and VEGF are elevated in melanoma patients harboring PTEN loss. In this study, the lipidation of autophagosome protein LC3 and autophagy in tumor cells, which can decrease T-cell priming and modulate resistance to T-cell-mediated apoptosis, is also inhibited due to the loss of PTEN protein and activation of PI3K.⁴⁶⁸ Consistent with these findings, previous studies have also found that inactivation of PI3Kδ could break Tregs-mediated tumor immune tolerance, resulting in the activation of CD8⁺ T-cell responses and subsequent tumor regression.^469–472 The Tregs T-cell receptor (TCR) downstream signaling, proliferation, and survival are dominantly dependent on PI3Kδ, but not PI3Kα or PI3Kβ.⁴⁷⁰ This potential adjuvant role for PI3Kδ inhibition in cancer immunotherapy was confirmed in a neoadjuvant, phase II clinical trial treatment-naive patients with resectable HNSCC.¹¹⁷ The inhibition of PI3Kδ by AMG319 decreased the number of tumor-infiltrating Tregs and activated intratumoral CD4⁺ and CD8⁺ T cells. However, the unfavorable safety profile should also be noticed, with frequent and severe grade 3/4 irAEs, probably driven by the systemic effect on Tregs in non-malignant tissues.¹¹⁷ Besides, as PI3K signaling is also essential in maintaining effector T-cell function,^473–475 a systemic inhibition of PI3Kδ impairs the function of CD8⁺ cytotoxic T lymphocytes, which antagonizes ICB therapy intending to boost the CD8⁺ T-cell response, counteracting any advantages brought by impairing intratumoral Tregs.⁴⁷⁶ The protocols of administrating PI3Kδ inhibitors were considered as an essential part. A modified treatment regimen with intermittent dosing of PI3Kδ inhibitors has shown a comparable antitumor efficacy while limiting toxicity.^115–117 In addition, given that PI3Kδ signaling might be required for signaling reactivation in exhausted T cells by ICB therapy,^477,478 sequential combination treatment might be more effective. The study of Isoyama et al.⁴⁷² confirmed that the combination protocol with anti-PD-1 mAb administrated first, followed by anti-PD-1 mAb plus PI3Kδ inhibitor induced the most effective and durable antitumor activity.

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Fig. 6

Impact of oncogenic signaling on tumor immune response. a Loss of PTEN protein function and improper PI3K activation inhibit efficient LC3 lipidation, which further promote resistance to T-cell-mediated killing by inhibiting autophagy. PTEN loss could also induce expression of immunosuppressive cytokines, including CCL12 and VEGF. b CDK4/6 inhibition enhances T-cell activation through the derepression of NFAT family proteins and their target genes, which encodes critical regulators of T-cell function. CDK4/6 inhibition could also induce Rb-mediated G1-arrest and promote the phenotypic and functional acquisition of immunologic T-cell memory. Besides, the PD-L1 protein stability is regulated by the CDK4-SPOP-FZR1 signaling pathway. Physiologically, PD-L1 protein stability is negatively regulated through phosphorylating its upstream physiological E3 ligase SPOP. This phosphorylation promotes SPOP binding to 14-3-3γ, which subsequently disrupts FZR1-mediated destruction of SPOP. The inhibition of CDK4/6 inhibits the phosphorylation of SPOP, thus promoting its degradation by FZR1, thus increasing PD-L1 protein levels. c Tumor-derived VEGF limits NF-κB activation in immature DCs, which in turn leads to defective functional maturation of DCs and insufficient induction of tumor immunity. VEGF could also impact the endothelial cells expression of immunological molecules. It decreases the expression of VCAM-1, which is important for the antitumor T cells adhesion and infiltration into tumors. Besides, VEGF also increases the expression of FAS ligand on endothelial cells, triggering apoptosis of T cells. VEGF also promotes the expansion of immune suppressive MDSCs, which further promotes the recruitment of Tregs. d EZH2 inhibition increases the production of CXCL9 and CXCL10, which are attractant cytokines promoting trafficking of T cells to tumor. Besides, EZH2 inhibition could selectively target intratumeral Tregs and reduce its immunosuppressive capacity. e In tumor cells, the ablation of LSD1 in cancer cells increases repetitive element expression, including ERVs, and decreases expression of RISC components. This leads to dsRNA stress and activation of type 1 interferon, which stimulates antitumor T-cell infiltration. In addition, inhibiting LSD1 in CD8⁺ T cells unleashes the transcription program mediated by TCF1, which is critical for the maintenance of the progenitor subset of intratumoral CD8⁺ T cells for persistent tumor control. dsRNA double-stranded RNA, ERVs endogenous retroviral elements, MDSCs myeloid-derived suppressor cells, RISC RNA-induced silencing complex, TCF1 T-cell factor 1, VCAM-1 vascular cell adhesion molecule-1

ICB and CDK4/6 inhibition

CDK4/6 is regarded as a promising target in treating LSCC when appropriate candidate patients are identified by the downstream functional assessment.³⁸ Recent studies have found that inhibition of CDK4/6 not only induces tumor cell-cycle arrest, but also increases T-cell inflammatory signature in tumors, which may act synergistically with ICB therapies^479,480 (Fig. ​(Fig.6b).6b). In mouse models, combining CDK4/6 inhibitors with PD-1 axis inhibitors resulted in significantly improved antitumor efficacy compared with either treatment alone.^480–483 The mechanisms of this synergistic effects have been under extensive studies recently. Goel et al.¹¹¹ found that the inhibition of CDK4/6 could increase the functional capacity of tumor cells to present antigens. Besides, CDK4/6 inhibitors could markedly and selectively reduce the immunosuppressive Tregs population.¹¹¹ This preference of CDK4/6 inhibitor may be attributed to the higher expression level of RB by Tregs (3.1-fold higher in Tregs than in CD8⁺ T cells,⁴⁸⁴) a key modulator of CDK4/6 pathway.⁴⁸⁵ The inhibition of CDK4/6 in tumor cells also increases PD-L1 protein levels,⁴⁸¹ which could be one of the mechanisms leading to the resistance of CDK4/6 inhibitor via evasion of immune checkpoints surveillance. This provides the rationale for the combination of PD-L1 blockade treatment and CDK4/6 inhibitors as a more potent antitumor treatment option. Properly timed and sequenced doses of CDK4/6 inhibitors could enhance T-cell activation⁴⁸³ and induce T-cell memory for maintaining long-term antitumor immunity.^486,487 Despite the great efficacy shown in preclinical models, some clinical trials co-administering CDK4/6 inhibitors and ICB therapy were halted because of severe toxicity (e.g., NCT02779751,^488,489 NCT04118036, NCT04075604). A different administrating method of CDK4/6 inhibitors has been proposed which may mitigate the toxicity risk.^486,487 The capacity of CDK4/6 inhibitors to promote T-cell memory gives rationales for using CDK4/6 inhibitors as a preconditioning tool, priming the T-cell pool before the application of ICB. In mouse models, preconditioning tumor-bearing mice with a CDK4/6 inhibitor significantly improved the efficacy of anti-PD-1 ICB therapy.⁴⁸⁶ Taken together, these results suggest CDK4/6 inhibition in combination with immunotherapy is a promising therapeutic strategy but still needs further investigation.

ICB and VEGF-VEGFR signaling blockade

With over 30 years of extensive research on VEGF, the biological role of VEGF has extended beyond its impact on neovascularization and angiogenesis, which also functions as an immunomodulator (Fig. ​(Fig.6c).6c). It has been shown that multiple immune cells could be influenced by VEGF, including DCs, T cells, Tregs, and MDSCs.⁴⁹⁰ Both in animal models and humans, the inhibition of VEGF could increase the number of tumor-infiltrating lymphocytes.^491,492 VEGF is also important for the expansion of the immunosuppressive MDSCs via its binding to VEGFR1, which further promotes an immunosuppressive microenvironment by de novo development of Tregs.⁴⁹³ In renal cell carcinoma, the inhibition of VEGF-VEGFR signaling by either mAbs or VEGF-TKIs could reverse MDSC-mediated immunosuppression.^494,495 This immunosuppressive profile of the VEGF/VEGFR axis is also confirmed by the fact that VEGF-A-VEGFR pathway blockade inhibits tumor-induced proliferation of Tregs.^496,497 Aberrant angiogenesis within the TME can mediate immune escape and reduce the efficacy of immunotherapy by hampering the delivery of drugs, oxygen, and effector T cells.^498,499 This tumor hypoxia promotes the recruitment of Tregs, which facilitates angiogenesis through excess production of VEGF.⁵⁰⁰ In hand with other tolerogenic leukocyte populations such as MDSCs⁵⁰¹ and pDCs⁵⁰² which also produce VEGF and support angiogenesis, this leads to further tumor tolerance and growth. During adaption to the hypoxic TME, tumor-infiltrating cytotoxic T cells are characterized by high glycolytic rates.^503,504 A recent study has found that hypoxia-inducible transcription factor 1α (HIF1α) is essential for the effector state in CD8⁺ T cells.⁵⁰⁵ VEGF-deficient CD8⁺ T cells showed lower efficiency on infiltrating tumors. An HIF1α/VEGF axis has been proposed in cytotoxic T cells to regulate tumor progression.⁵⁰⁵ The above-mentioned findings provide further evidence for the ongoing clinical evaluation of combined immunotherapies and antiangiogenic approaches. Currently, the only approved combination therapy of immunotherapy and antiangiogenic approach for NSCLC is the PD-L1 inhibitor atezolizumab in combination with bevacizumab, paclitaxel, and carboplatin for the first-line treatment of patients with metastatic non-squamous NSCLC with no EGFR or ALK genomic tumor aberrations.⁵⁰⁶ AK112, a tetrameric bispecific antibody targeting PD-1 and VEGF, has shown in a phase I dose-escalation study with a manageable safety profile.⁵⁰⁷ It is currently being evaluated in a phase II clinical trial for stage IIIB/C or IV NSCLC (NCT04736823).

This study was supported by National Key Research and Development Program of China (2021YFC2500903, 2021YFC2500905), Natural Science Foundation of Shanghai (22ZR1439200), National Natural Science Foundation of China (82072557, 81871882), Shanghai Municipal Commission of Health and Family Planning Outstanding Academic Leaders Training Program (2017BR055), and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant (20172005). Some icons or graphic elements in Figs. ​Figs.2,2, ​,3,3, ​,4,4, ​,5,5, and ​and66 are adapted from BioRender.com (2022), retrieved from https://app.biorender.com/. Final schematic illustrations were created and integrated by our original design.