Receptor tyrosine kinase-like orphan receptor 1: A novel antitumor target in gastrointestinal cancers

Abstract

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a member of the type I receptor tyrosine kinase family. ROR1 is pivotal in embryonic development and cancer, and serves as a biomarker and therapeutic target. It has soluble and membrane-bound subtypes, with the latter highly expressed in tumors. ROR1 is conserved throughout evolution and may play a role in the development of gastrointestinal cancer through multiple signaling pathways and molecular mechanisms. Studies suggest that overexpression of ROR1 may increase tumor invasiveness and metastasis. Additionally, ROR1 may regulate the cell cycle, stem cell characteristics, and interact with other signaling pathways to affect cancer progression. This review explores the structure, expression and role of ROR1 in the development of gastrointestinal cancers. It discusses current antitumor strategies, outlining challenges and prospects for treatment.

Key Words: Receptor tyrosine kinase-like orphan receptor 1; Gastrointestinal cancers; Therapeutic target; Molecular mechanisms; Antitumor strategies

Core Tip: Delve into a comprehensive review spotlighting receptor tyrosine kinase-like orphan receptor 1 (ROR1) pivotal involvement in gastrointestinal cancer advancement and its promising prospects as an anti-tumor remedy. Explore pivotal cellular pathways governing ROR1 regulation and pertinent insights into existing commercial therapeutic offerings. Emphasizing ROR1’s crucial role in gastrointestinal cancer treatment, this review offers a nuanced blend of historical context and scientific insight, illuminating the dynamic landscape of this evolving field.

INTRODUCTION

Receptor tyrosine kinase-like orphan receptor (ROR) 1 and ROR2 belong to the ROR subfamily of type I receptor tyrosine kinase (RTK) family. They were first identified in human neuroblastoma cells in 1992 and are termed orphan receptors as their ligands are not known[1-3]. The two proteins share 58% sequence identity. ROR1 has recently been shown to be involved in embryonic development and cancer. It is recognized as a diagnostic biomarker and is a research hotspot for the targeted treatment of various malignancies. ROR1 has two subtypes; an intact membrane receptor and a truncated variant. The truncated versions include a membrane-bound form that lacks an extracellular domain and a soluble form that contains only the extracellular structure[4]. Expression of the soluble isoform does not increase in cancer patients, showing minimal or undetectable levels in the serum that are not dependent on disease progression or severity, whereas expression of the intact membrane-bound isoform increases significantly[5].

ROR1 proteins are found in a variety of species. Drosophila melanogaster has a protein called Dror, which resembles an RTK. The kinase and structural extracellular domains of Dror display 61% and 36% sequence identity, respectively, with the corresponding domains of human ROR1[6], while human and mouse ROR1 are 97% identical. This evolutionary conservation of ROR family proteins indicates the importance of their physiological functions during embryonic and organ development. Many recent studies on ROR1 have demonstrated its aberrant expression in various forms of cancer. Abnormal expression of ROR1 was first observed in chronic lymphocytic leukemia (CLL)[2,7]. Although specific disease-related mutations have yet to be identified in ROR1, it has become a widely studied therapeutic target for localized ischemia, diabetes, and malignancies[8-11].

Gastrointestinal cancers, including liver, gastric, colorectal, and pancreatic cancer, are some of the most frequently diagnosed tumors and contribute significantly to cancer-associated mortality in China and worldwide. Overexpression of ROR1 is seen in many gastrointestinal cancers, where it is linked with poor prognosis, indicating the essential roles of ROR1 in these tumors. This paper reviews the structure and expression patterns of ROR1 in gastrointestinal cancers, as well as its involvement in cancer development. Current antitumor strategies for gastrointestinal cancers involving ROR1 are discussed and the challenges and prospects in treating these diseases are summarized.

STRUCTURE AND EXPRESSION PATTERNS OF ROR1

ROR1 is a type I transmembrane protein, 937 residues in length, with a molecular weight of approximately 105 kDa. The protein includes extracellular, transmembrane, and intracellular regions (Figure 1). The extracellular region includes several extracellular domains, specifically, immunoglobulin-like (Ig-like), frizzled (FZD), and Kringle (KD) domains. FZD is a cell membrane receptor for the secreted glycoprotein Wnt. It contains a cysteine-rich domain located at the N terminus outside the cell that can bind to Wnt. The FZD domain regulates nonclassical Wnt signaling through interaction with its ligand, Wnt5a, while KD mediates interactions between ROR1 and other receptors, including ROR2. The intracellular domains include three domain types; one tyrosine kinase (TK)-like and two serine/threonine-rich domains, together with a proline-rich domain. The serine/threonine-rich domains bind to bridging proteins such as 14-3-3ζ to prevent apoptosis[12], while the proline-rich domain binds to Src homology 3 (SH3) domains in various proteins, such as hematopoietic lineage cell-specific protein 1 (HS1), dedicator of cytokinesis protein 2 (DOCK2), and cortactin, to promote cell migration and proliferation[13-15]. However, although ROR1 is an RTK, the function of its TK domain remains essentially unknown.

Figure 1 Diagram of the receptor tyrosine kinase-like orphan receptor 1 domain structure. IG: Ig-like domain; FZD: Frizzled domain; KD: Kringle domain; TKD: Tyrosine kinase domain; S/T: Serine/Threonine-rich domain; PRD: Proline-rich domain.

ROR1 expression is primarily limited to embryonic development with low levels seen in postnatal tissue. Nevertheless, some normal tissues, including adipose tissue, endocrine glands, and the gastrointestinal tract, display high levels of ROR1 expression[16,17]. ROR1 knockout is fatal in the embryo, providing evidence of its crucial role in embryonic development[18]. In contrast to the relatively low or minimal expression observed in normal tissues, ROR1 expression increased markedly in a variety of hematological cancers and solid tumors, including CLL, breast cancer, melanoma, and gastrointestinal cancers, such as colorectal cancers and pancreatic ductal adenocarcinomas[19-22]. This feature suggests that ROR1 may be a promising candidate for cancer therapy.

REGULATORY MECHANISMS OF SIGNALING PATHWAYS INVOLVED IN ROR1

Aberrant expression of ROR1 protein in CLL cells was identified using monoclonal antibodies[19]. These authors also found that Wnt5a bound specifically to ROR1 to promote cancer growth and survival, thus ending the orphan status of ROR1. Wnt5a has been shown to activate the non-classical Wnt pathway through interaction with the FZD domain of ROR1[18,23], indicating the potential importance of ROR1 in various biological processes through non-canonical Wnt signaling. Wnt5a is also involved in phosphorylation of the Nuclear Factor-kappa B (NF-κB) subunit p65, activating NF-κB signaling in cancer cells, and thus promoting tumorigenesis, the epithelial-mesenchymal transition (EMT), and metastasis. NF-κB signaling is also closely linked with both inflammation and immune regulation, and shows significant activation in various tumor types.

The Wnt5a pathway is a vital component of the Wnt signaling pathway family and plays a significant role in cancer progression. It affects cell behaviors such as proliferation, differentiation, and migration, which can impact cancer development. Dysregulation of this pathway has been linked to various cancers. By regulating cell cycle and migration, it restricts cancer cell growth and invasion. Additionally, it may impact cancer stem cell characteristics and immune responses within the tumor microenvironment. Furthermore, it interacts with neighboring tissues, promotes angiogenesis, and induces inflammation. Its functions differ among various types of cancer, leading to investigations into its regulatory mechanisms as potential therapeutic targets.

The FZD gene family includes seven transmembrane proteins that are receptors for Wnt ligands. FZD5 appears to be the receptor for Wnt5a. As shown in Figure 2, Wnt5a can activate both FZD5 and ROR1, leading to activation of dishevelled 2/3 (Dvl2/3) and protein kinase B (Akt) phosphorylation. This leads to Akt-mediated phosphorylation of IκB kinase α (IKKα) and activation of IκB kinase (IKK), inducing Inhibitor of kappa B α (IκBα) degradation and NF-κB subunit p65 phosphorylation. After phosphorylation, p65 translocates to the nucleus where it promotes the transcription of various genes including Cyclin D1, cellular myelocytomatosis (c-Myc), matrix metalloproteinase-9, and Vascular Endothelial Growth Factor, as well as Wnt5a to induce an autonomous feedback loop. Continued stimulation of the ROR1/Akt/p65 pathway within this loop further promotes the production of proinflammatory factors such as interleukin-6 (IL-6) and chemokines such as CCL2[24]. Further research found a positive correlation between ROR1 expression and increased transcription of Yes-associated protein/Transcriptional co-Activator with PDZ-binding motif (YAP/TAZ), promoting both tumorigenesis and chemotherapy resistance[25,26]. Complexation of Wnt5a with ROR1/FZD promotes binding between G protein subunit alpha 12/13 (Gα12/13) and the transforming protein Ras homolog gene family, member A (RhoA), inhibiting the activity of large tumor suppressor kinase 1/2 (Lats1/2) and stimulating YAP/TAZ dephosphorylation and nuclear translocation. Gα12/13 belongs to the gα protein family, where it differs from other members of the family, G protein subunit alpha s (Gαs), G protein subunit alpha i (Gαi), and G protein subunit alpha q (Gαq), and modulates cytoskeletal reorganization and morphological changes in the cell by activating the small Guanosine Triphosphatase (GTPase) Rho. Gα12/13 is also involved in processes such as cell proliferation, migration, and apoptosis. Nuclear translocation of YAP/TAZ, together with transcription enhanced association domain (TEAD), increases the transcription of genes associated with stem cell renewal, proliferation, and tumorigenesis. The upregulation of ROR1 expression is, in turn, stimulated by increased YAP/TAZ transcription.

Figure 2 Signaling pathways regulated by receptor tyrosine kinase-like orphan receptor 1. Dvl2/3: Dishevelled 2/3; Akt: Protein kinase B; Iκκα: IκB kinase α; IκBα: Inhibitor of kappa B α; p65: Nuclear factor NF-kappa-B p65 subunit; RhoA: Ras homolog gene family, member A; Gα12/13: G protein subunit alpha 12/13; Lats1/2: Large tumor suppressor kinase 1/2; YAP/TAZ: Yes-associated protein/Transcriptional co-Activator with PDZ-binding motif. Binding of Wnt5a to receptor tyrosine kinase-like orphan receptor 1 (ROR1) induces Dvl2/3 activation and Akt phosphorylation, leading to phosphorylation of Iκκα, activation of the IκB kinase complex, degradation of IκBα, and p65 phosphorylation. Subsequently, p65 is transferred to the nucleus where it promotes the transcription of specific genes, including Wnt5a, producing an autonomous feedback loop. Binding between Wnt5a and the ROR1/frizzled complex leads to activation of RhoA via Gα12/13. This activation inhibits Lats1/2 activity, leading to the dephosphorylation and nuclear translocation of YAP/TAZ. The binding of YAP/TAZ to transcription enhanced association domain increases the transcription of genes linked to tumorigenesis and stemness while increased levels of YAP/TAZ enhance ROR1 expression.

ROLE OF ROR1 IN GASTROINTESTINAL CANCERS

The mechanisms of ROR1 action in diverse malignancies, encompassing gastrointestinal tumors such as liver, gastric, colorectal, and pancreatic cancer, exhibit heterogeneity. Distinct signaling pathways or processes influenced by ROR1 may be implicated in each specific cancer type. Therefore, it is necessary to discuss the function of ROR1 in these cancers individually.

ROR1 and liver cancer

Liver cancer is one of the six most common cancers worldwide and falls into three main types, namely, hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma, and mixed cell carcinoma[27]. HCC is the most prevalent and lethal variety, representing approximately 90% of all liver cancers. Liver tumors may also be derived from metastasis of other malignancies, such as lung tumors. Due to its high mortality rate and ease of metastasis, HCC is a subject of significant interest in cancer research[28].

A study by Cetin et al[29] assessed ROR1 in HCC, demonstrating its presence in both human and mouse cells, without limitation to metastatic or mesenchymal cell lines. Induction of EMT by transforming growth factor-β (TGF-β) reduced ROR1 levels, while ROR1 knockdown inhibited both the proliferation and migration of HCC cells and increased resistance to apoptosis. Chemotherapeutic agent uptake was modified in ROR1 knockdown epithelial-type HCC cells, leading to resistance to chemotherapy-induced apoptosis. ROR1 has been shown to promote HCC progression through the modulation of various pathways, and changes in ROR1 expression may be useful in diagnosis and prognostic prediction. Meng et al[30] reported that ROR1 expression is substantially increased in acid-treated cancer cells and significantly promotes invasion and migration of HCC. ROR1 knockdown through siRNA successfully inhibited acid-induced tumor cell migration, invasion, and EMT. Nevertheless, the underlying regulatory actions of ROR1 in HCC are not yet clear.

ROR1 and gastric cancer

Gastric cancer (GC) is a heterogeneous disease with a high mortality rate. It is known that infection with Helicobacter pylori is both necessary and insufficient for the development of GC[31] which appears to involve a multifactorial and multistep pathogenesis[32].

There is limited information on ROR1 in GC. Kotoh et al[33] found an association between Wnt5a and GC invasion and metastasis, showing that Wnt5a bound to FZD/ROR1 and was linked to various signaling pathways including β-catenin-TCF/LEF, Dishevelled–RhoA–Rho-associated protein kinase (DVL–RhoA–ROCK), and mitogen-activated protein kinase kinase kinase 7–nuclear factor-B (MAP3K7–NF-κB) in specific contexts. ROR1 also associates with c-Src, promoting its phosphorylation. C-Src is a non-RTK and known oncogene that has been linked to the development of numerous human cancers, including colon, gastric, lung, breast, and prostate cancers. Once activated, c-Src can regulate a variety of processes, both normal and cancer-associated, including cell survival, proliferation, motility, differentiation, and angiogenesis[34,35].

ROR1 and colorectal cancer

Colorectal cancer (CRC) is among the most prevalent cancer types globally, ranking fifth in incidence in China. Advanced CRC is associated with both recurrence and drug resistance, reducing patient survival. Thus, investigation of the mechanisms underlying CRC is important to improve survival and develop new drugs. Zhou and colleagues investigated expression of ROR1 in CRC[36], observing markedly increased levels in CRC tissues and in approximately 94% of patients with CRC. However, the molecular mechanisms underlying these increases remain unknown.

Wnt signaling is closely associated with regulation of morphogenesis during embryonic development and repair processes. The Wnt ligands are key to these pathways, and are known to bind different receptors, including proteins of the FZD family and ROR1. The interactions trigger downstream cascades involving multiple pathways to influence the arrangement of the cytoskeleton, transcription of genes associated with proliferation and cell growth, and the behavior of intracellular organelles. Dysregulated Wnt signaling has been linked to various malignancies, including CRC, through the modulation of cells associated with the tumor microenvironment (TME)[37].

ROR1 and pancreatic cancer

Pancreatic cancer is an especially lethal form of cancer. Despite significant progress in elucidating its molecular pathology, the prognosis remains extremely poor, mostly due to treatment resistance and the formation of distant metastases in the early stages of the disease[28]. Therefore, uncovering the underlying molecular mechanisms of pancreatic cancer to create new targeted therapeutic strategies remains a pressing research priority.

Yamazaki et al[38] demonstrated that ROR1 promotes the proliferation of pancreatic ductal adenocarcinoma (PDAC) by activating E2F via c-Myc and inducing expression of aurora kinase B. It was also found that transcription of ROR1 was dependent on the binding of YAP/BRD4 to its promoter region. Prevention of this binding reduced ROR1 expression and inhibited PDAC growth. These findings suggest that increased levels of ROR1 are closely involved in tumorigenesis, highlighting its importance in PDAC progression and its potential as a target for treating the cancer.

ANTITUMOR STRATEGIES TARGETING ROR1 IN GASTROINTESTINAL CANCERS

There has been an increased focus on ROR1 as a potential antitumor target for gastrointestinal cancers, involving various therapeutic approaches and ongoing clinical trials and preclinical studies. These include the use of monoclonal antibodies (mAbs), antibody–drug conjugates (ADCs), bispecific antibodies, small molecule inhibitors, and chimeric antigen receptor (CAR)-T cell therapies (Table 1).

Table 1 Receptor tyrosine kinase-like orphan receptor 1-targetd therapies in clinical trials or preclinical studies.

Therapy types	Therapy drugs	Indications	ClinicalTrials.gov Identifier/Ref.
mAb	Zilovertamab	Gastric cancer Pancreatic cancer	[42]
ADC	VLS-101	Solid tumor	NCT04504916
	LCB71		NCT05279300
	NBE-002		NCT04441099
	miR-29b		[47]
	OSU-2S		[48]
Bispecific antibody	R11×v9-BiAb	Solid tumor	[50]
	NVG-111		NCT04763083
	SFG.ROR1-BiTE	Pancreatic cancer	[51]
Small-molecule inhibitors	KAN0439834	Pancreatic cancer	[54]
	KAN04415711C		[55]
	miR-27b-3p	Gastric cancer	[57]

mAb: Monoclonal antibodies; ADC: Antibody drug conjugate.

Anti-ROR1 monoclonal antibodies

The application of anti-ROR1 mAbs to block binding between the receptor and the Wnt5a ligand prevents activation of downstream signaling and activates the immune system to promote removal of tumor cells[39]. Zilovertamab, also known as cirmtuzumab or UC-961, is an mAb targeting ROR1. Zilovertamab binding to ROR1 blocks Wnt5a signaling, thus inhibiting tumorigenic behavior, such as cell growth and survival, and inducing differentiation. This mAb has shown significant antitumor efficacy against both hematological and solid malignancies, such as GC and pancreatic cancer, in Phase I clinical trials, and is currently the only antibody on the market that has undergone clinical evaluations[40]. A phase I/II trial of a combination of zilovertamab and paclitaxel for treating breast cancer is currently underway.

Chen et al[24] reported that the Wnt5a/ROR1 axis can activate the NF-κB pathway in tumor cells, resulting in the production of interleukin-6 and signal transducer and activator of transcription 3 phosphorylation. Zilovertamab has the potential to inhibit these processes. According to Hasan et al[15], Wnt5a promotes complexation between ROR1 and DOCK2, leading to the activation of Rac1/2. Silencing of DOCK2 specifically reduces Wnt5a-mediated activation of Rac1/2 and tumor cell proliferation. The proline-rich domain (PRD) of ROR1 has been implicated in the Wnt5a interaction with DOCK2 and activation of Rac1/2 in MEC1 CLL cells. This effect is blocked by zilovertamab, which also has the advantages of having a long half-life in the plasma and no dose-limiting toxicity. The use of this antibody showed reductions in the activities of RhoA and HS1, while transcriptome analysis indicates reductions in the expression of stemness genes in CLL in vivo. It is apparent that zilovertamab is both safe and effective for the inhibition of ROR1 in various tumor cells[40].

Antibody-drug conjugates

ADCs involve linkage between mAbs and small cytotoxic molecules via specific chemical linkers. ADCs targeting ROR1 are able to transfer cytotoxic agents and regulatory RNAs to ROR1-expressing tumor cells.

VLS-101 (MK-2140) is an ADC consisting of a lead antibody to effectively target ROR1, for treating hematological malignancies and solid tumors. VLS-101 represents a combination of zilovertamab, a lysyl iminohexene-valine-citrulline p-aminobenzoate linker, and a cytotoxic agent monomethyl auristatin E (MMAE) that targets microtubules. Within cells, MMAE is released from the linker to block microtubule polymerization, thus adversely affecting mitosis and cell division[41]. The drug also safely promotes tumor regression in patient-derived xenograft models previously found to be resistant to CAR-T cell, ibrutinib, and/or venetoclax treatment[42]. These findings provide valuable insights into the increasing clinical demand for VLS-101 in gastrointestinal cancers.

LCB71 (CS5001) is an ADC based on an mAb that targets ROR1. LCB71 has a distinctive design involving a unique β-glucosidic acid linker and a pyrrolobenzodiazepine (PBD) pre-toxin dimer. Both the linker and the pretoxin are cleaved by lysosomal β-glucuronidase, an enzyme significantly overexpressed in many types of cancer cells. Thus, LCB71 can eliminate cancerous cells after arrival at the tumor site and cleavage of the linker to release the PBD pre-toxin, which is subsequently activated within the tumor cell. The dual control mechanism of the linker plus precursor toxin resolves typical toxicity issues associated with traditional PBD loads, resulting in an improved safety profile. LCB71 has shown notable dose-dependent antitumor activity in several xenograft tumor models of condylomatous lymphoma and breast cancer and is thus a promising drug candidate with potential for precision therapy in both hematological and solid malignant tumors expressing high levels of ROR1. The ADC NBE-002 is composed of a humanized antibody, huXBR1-402, conjugated to the anthracycline derivative, PNU-159682. HuXBR1-402 is a chimeric rabbit/human mAb identified from a rabbit antibody library using phage display. NBE-002 has been shown to be effective in xenograft models of various human tumors[43]. The drug is currently in a clinical Phase I study. Chiang et al[44] connected an anti-ROR1 mAb to a regulatory miRNA (miR-29b). This ADC treatment reduced the expression of the miR-29 targets, DNA methyltransferase (DNMT) 1 and DNMT3A, in cells expressing ROR1. It also led to alterations in global DNA methylation patterns, downregulation of SP1, and upregulation of p21. Another ADC used an mAb against ROR1 linked to nanoparticles containing OSU-2S, a sphingosine analog with anti-tumor capabilities, that activated protein phosphatase 2A and induced SHP1 phosphorylation and nuclear translocation[45], resulting in apoptosis.

Bispecific antibody

Bispecific antibodies (BiAbs) are synthetic antibodies created through cell fusion or recombinant DNA technology. They are formed by two single-chain antibody fragments, or single-chain fragment variable (scFvs), together with different variable regions composed of both heavy and light chains and a common constant region, or Fc. Bispecific T-cell conjugates (BiTes) are fusion proteins composed of two separate scFvs without an Fc. One arm can bind to the Kringle domain on ROR1 while the other interacts with the CD3 subunit of the T cell receptor, thus recruiting polyclonal T cells to the site of the tumor. BiTEs do not rely on antigen presentation and can activate T-cell binding, resulting in the killing of neighboring target cells. Both BiAbs and BiTes can bind simultaneously to two different antigens or epitopes on the same antigen. Due to their specificity and bifunctionality, the use of BiAbs and BiTes is a trending research topic in antibody engineering and has a broad range of applications in tumor therapy and autoimmune diseases.

Qi et al[46] co-conjugated multiple anti-ROR1 single-chain antibodies to a single-chain antibody against CD3 with glycosylated IgG1-Fc, generating various BiAbs. They found that targeting scFv using ROR1 with the proximal epitope R11 of the membrane was the most effective one for recruiting T cells to ROR1-positive solid cancer cell lines. This BiAb exhibited promising antitumor activity both in vitro and in vivo. In a similar investigation, Wang et al[47] also used a BiAb composed of an scFv and Fc that targeted both ROR1 and CD3; this was termed R11×v9-biAb. When tested on multiple ROR1-positive solid cancer cell lines, the observed cytotoxicity was found to be dependent on the R11×v9-biAb dosage. The BiAb targeted ROR1-positive cancerous cells, induced tumor cell death, and recruited significant numbers of CD4+ and CD8+ T cells to the tumor through the secretion of T-cell-derived proinflammatory factors and promoting perforin production by granzyme B and CD8+ T cells.

NVG-111 is a unique humanized BiTE recognizing both ROR1 and CD3, and is composed of tandem scFvs against ROR1 and CD3. NVG-111 is the first BiAb that utilized the inherent cytotoxicity of T cells. Ongoing clinical trials of NVG-111 have shown promising results in both CLL and solid tumors, with favorable cytotoxicity[48]. Another bispecific T-cell splicer, SFG. ROR1-BITE, includes scFvs targeting the cysteine-rich coiled-coil structural domain of ROR1 and CD3, and showed strong cytotoxicity against pancreatic cancer cells expressing ROR1 at low concentrations, as well as significant tumor reduction in a mouse model of pancreatic tumors[49].

Small-molecule inhibitors of ROR1

The lack of clearly defined kinase activity in ROR1 has limited the identification of small-molecule inhibitors that could target this activity. Although some catalytic activity was observed in immunoprecipitated ROR1 suggesting the possibility of autophosphorylation in vivo, this was found to be negligible[50,51].

Nevertheless, Hojjat-Farsangi et al[52] identified a promising inhibitor, KAN0439834, against ROR1 by screening a library of 110000 small molecules. This compound targeted the TK domain, preventing phosphorylation of ROR1 by Wnt5a and thus blocking the activation of downstream proteins such as Src, Akt, protein kinase C, and MAPK. Further research demonstrated that KAN0439834 was more effective in inducing apoptosis in cancer cells than mAbs with a potency equivalent to that of venetoclax, an inhibitor of Bcl-2 used for the treatment of patients with CLL. Clinical trials have also shown that combinations of KAN0439834 with erlotinib and ibrutinib, small-molecule inhibitors of epidermal growth factor receptor and Bruton's tyrosine kinase, act synergistically in the treatment of patients with pancreatic cancer[53]. The same group screened KAN04415711C, a second-generation compound targeting ROR1, which was found to be effective against tumors in a zebrafish model[54]. KAN04415711C was also effective in vitro when combined with venetoclax. Its mechanism of action, however, requires further investigation.

ROR1-targeted CAR-T cell therapy

CAR-T therapy involves the transfer of structural domains that specifically recognize antigens and genetic material promoting T cell activation signals into T-cells. This enables the T cells to target the specific antigens on the surfaces of cancer cells, leading to the release of cytotoxic compounds such as perforin and granzyme B. Cytokines may also be released to recruit endogenous immune cells for cotreatment. Additionally, the formation of memory T cells can establish a long-lasting antitumor mechanism in vivo[55-57].

ROR1 is currently considered a promising target in the design of CAR-T cell therapy. Hudecek et al[58] and Hudecek et al[59] designed a range of CAR-T cells targeting ROR1, featuring a concise “hinge-only” extracellular spacer, that was found to be highly effective in destroying ROR1-positive tumor cells and inducing T cell effector functions. The safety of this therapy is currently being evaluated due to concerns about cross-reaction with normal tissues by the scFv[60,61]. Lee et al[62] developed two CAR-T cell constructs targeting ROR1 by utilization of the antigen-recognition domains of zilovertamab and different hinge regions on the antibody. It was found that the construct with a truncated IgG4 hinge fragment was the most successful in terms of expression and in vitro functionality. Additionally, it effectively controlled tumor growth in xenograft mouse models with no significant toxicity. This study demonstrates the potential of Zilovertamab-derived ROR1 CAR-T for the clinical treatment of solid tumors.

The vast majority of candidate CAR targets in solid tumors, such as HER2, GD2, and mesothelin, are co-expressed in a variety of normal tissues, making off-tumor toxicity a significant risk for immunotherapies targeting these molecules. This makes it crucial to study the toxicity of ROR1 targeted therapy for gastrointestinal tumors, as ROR1 is no exception. SNIP CARs provide a solution to CAR-T therapy toxicities by utilizing a regulated protease-based system controlled by an FDA-approved small molecule. They offer improved potency and safety in various models, enabling remote control over CAR activity for treating solid tumors[63].

A study by Srivastava and colleagues[64] found that ROR1 CAR-T therapy led to fatal bone marrow failure in mice due to the recognition of ROR1-positive stromal cells. To enhance selectivity, they integrated synthetic Notch (synNotch) receptors specifically recognizing epithelial cell adhesion molecule or B7-H3 into T cells. These receptors were expressed on ROR1-positive cancer cells but not on stromal cells expressing ROR1. This induced a selective ROR1 CAR-T effect, leading to successful tumor ablation without toxicity in normal ROR1-positive cells. The study demonstrated that the use of synNotch receptors for combined antigen sensing can prevent CAR-T-cell-mediated lethal toxicity to normal tissue, provided that the tumor and normal tissue were spatially separated. However, if they are highly colocalized at the same site, this approach may not be effective. Moreover, the same group further discovered that ROR1 CAR-T cells showed poor tumor infiltration and malfunctioned. The inclusion of oxaliplatin, however, in the regimen allowed the recruitment of T cells by activated tumor macrophages[65], improving CAR-T-cell infiltration to modify the TME and increasing the sensitivity of the tumor to immunotherapy. Thus, the ROR1 CAR-T combination therapy provides an alternative strategy for the enhancement of CAR-T cell efficacy in clinical settings.

Some metabolites and small-molecule drugs can boost the anticancer actions of CAR-T cells. It was found that some short-chain fatty acids, such as butyrate and valerate, could enhance the actions of cytotoxic T lymphocytes and CAR-T cells through both epigenetic and metabolic means[66]. Furthermore, SD-208, a kinase inhibitor, could enhance the cytolytic activity, cytokine production, viability, and proliferation of ROR1-CAR T cells by blocking TGF-β receptor signaling[67]. These findings underscore the significance of novel combination therapies in enhancing the efficacy of ROR1 CAR T cells for treating gastrointestinal cancers.

CONCLUSION

ROR1 is a potentially effective therapeutic target for various gastrointestinal cancers. However, high expression of ROR1 on the surface of normal tissues poses potential risks for off-target tumor toxicity. Therefore, the use of treatments targeting ROR1 requires monitoring of potential toxicity to healthy tissues to ensure safety. The effectiveness of mAbs, ADCs, or BiTEs against ROR1 on solid tumors, including gastrointestinal cancers, may be restricted due to the characteristics of the TME and tumor heterogeneity. Therefore, if long-term localized expression of anti-ROR1 BiTEs or mAbs can be induced within tumors, this could recruit and activate tumor-infiltrating lymphocytes for effective antitumor effects. Oncolytic viruses can serve as vectors for delivering ROR1 BiTEs or mAbs, potentially offering a solution to this problem. Our group has constructed a novel oncolytic adenovirus that delivers anti-ROR1-BiTE, which has demonstrated efficient inhibition of the growth of tumor cells in a mouse xenograft model. This highlights the importance of the combination of oncolytic virotherapy with ROR1 CAR T cell therapy. Overall, the in-depth research into the use of treatments targeting ROR1 suggests that better strategies for the clinical treatment of gastrointestinal cancer will be developed in the future.