ABIN-1 is a key regulator in RIPK1-dependent apoptosis (RDA) and necroptosis, and ABIN-1 deficiency potentiates necroptosis-based cancer therapy in colorectal cancer

ABIN-1, also called TNIP1, is an ubiquitin-binding protein that serves an important role in suppressing RIPK1-independent apoptosis, necroptosis, and NF-κB activation. However, the involvement of ABIN-1 in the regulation of RIPK1-dependent apoptosis (RDA) is unknown. In this study, we found that poly(I:C) + TAK1 inhibitor 5Z-7-oxozeaenol (P5) concurrently induces RDA and necroptosis in Abin-1−/−, but not in Abin-1+/+ mouse embryonic fibroblasts (MEFs). Upon P5 stimulation, cells initially die by necroptosis and subsequently by RDA. Furthermore, we explored the therapeutic effect of ABIN-1 deficiency in necroptosis-based cancer therapy in colorectal cancer (CRC). We found that poly(I:C) + 5Z-7-oxozeaenol + IDN-6556 (P5I) yields a robust pro-necroptosis response, and ABIN-1 deficiency additionally enhances this P5I-induced necroptosis. Moreover, phase I/II cIAP inhibitor birinapant with clinical caspase inhibitor IDN-6556 (BI) alone and 5-fluorouracil with IDN-6556 (FI) alone are sufficient to induce necroptotic cell death in CRC cells by promoting auto-secretion of tumor necrosis factor (TNF); ABIN-1 deficiency amplifies the BI- or FI-induced necroptosis. Two independent xenograft experiments using HT-29 or COLO205 cells show that both BI and P5I remarkably inhibit tumor growth via necroptosis activation. For poly(I:C)-induced cell death, the sensitizing effect of ABIN-1 deficiency on cell death may be attributed to increased expression of TLR3. In TNF-induced necroptosis, ABIN-1 deficiency increases TNF-induced RIPK1 polyubiquitination by reducing the recruitment of ubiquitin-editing enzyme A20 to the TNFR1 signaling complex and induces more TNF secretion in CRC cells upon pro-necroptosis stimulation. With this combined data, ABIN-1 deficiency promotes greater sensitization of CRC cells to necroptosis.

In addition to TNF-induced necroptosis, another common necroptotic cell death is Toll-like receptor 3 (TLR3)induced necroptosis triggered by poly(I:C) and caspase inhibitors. Specifically, the engagement of TLR3 induces the oligomerization of the adaptor protein TRIF that in turn recruits cIAPs, caspase-8/FADD, and RIPK1 leading to either apoptosis or necroptosis 13 If caspase-8 is inhibited by caspase inhibitors, RIPK1 recruits RIPK3 and MLKL to necroptosis; however, if caspase-8 is active, cells mostly undergo apoptosis 13,14 . Currently, it is unclear if suppression of TAK1 can promote RDA upon poly(I:C) stimulation.
A20-binding inhibitor of NF-κB1 (ABIN-1), also called TNIP1, is an ubiquitin-binding protein with a UBAN domain, which serves an important role in suppressing apoptosis, necroptosis, and NF-κB activation 15,16 . ABIN-1 deficiency also enhances innate immunity and antiviral responses 17 . Specific haplotypes or dysfunction in Abin-1 increase the risk of suffering autoimmune diseases 18 . Previously, we have proven that ABIN-1 is recruited to the TNFR1 signaling complex (TNF-RSC) in an M1 ubiquitination-dependent manner to facilitate the recruitment of the ubiquitin-editing enzyme A20. A20 functions as a K63 deubiquitinase of RIPK1 and deficiency of A20 thus promotes necroptosis by activating RIPK1 and its pro-necroptotic partner RIPK3 16,19 . Abin-1 −/− mice die at embryonic day 18.5 from liver damage that can be rescued by Ripk1 D138N kinase-dead mutation or by Ripk3 deficiency 16 . ABIN-1 deficiency can transform TNF + cyclohyximide (TC)-induced RIPK1-independent apoptosis into both RIPK1-independent apoptosis and necroptosis in mouse embryonic fibroblasts (MEFs) 16 , However, it is unknown whether ABIN-1 deficiency also participates in the regulation of RDA. Moreover, further investigation is necessary to understand whether targeting ABIN-1 can be applied to improve necroptosis-based cancer therapy.
Necroptosis-based cancer therapy is an alternative option when pro-apoptosis chemotherapy fails due to drug resistance. Many proposed mechanisms of cancer therapy failure are disrupted apoptosis machinery, strengthened prosurvival signals, increased expression of therapeutic targets, activation of compensatory pathways, high molecular heterogeneity in tumor cells, upregulation of drug transporters, and multidrug resistance 20,21 . Among these reasons, disrupted apoptosis machinery due to caspase inhibition and the defect is a critical factor in intrinsic and acquired chemotherapy drug resistance. Therefore, necroptosis may kill cancer cells with defective and inhibited caspases 22 . In addition, necroptotic cancer cells release many damage-associated molecular patterns (DAMPs) and tumor antigens to activate dendritic cells, which in turn activates CD8 + T cells and antitumor immunity 23 . Notably, RIPK1 and NF-κB signaling in necroptotic cells is critical for CD8 + T-cell crosspriming 24 . In the past few years, some studies have illustrated the potential therapeutic effect of necroptosisbased cancer therapy in tumor models 22,25 . A study by Xie et al. showed that inhibition of Aurora kinase A (a negative regulator of RIPK1-RIPK3-MLKL necrosome) by CCT137690-induced necroptosis and suppressed the growth and progression of both subcutaneous and orthotopic pancreatic tumors in mice 26 . 5-FU with pancaspase inhibitor IDN-7314-induced TNF-dependent necroptosis in colon cancer cells showed a better suppressive effect in tumor growth in HT-29 xenograft model compared to 5-fluorouracil alone. Moreover, around 50% of tumor samples from colon cancer patients were more sensitive to necroptosis inducer 5-FU + zVAD than apoptosis inducer 5-FU 27 . Poly(I:C) + zVAD also served as a necroptosis inducer and inhibit tumor growth in colon cancer xenograft model 28 .
Considering colorectal cancer (CRC) cells are more responsive to pro-necroptosis stimuli compared with other cancer types, it would be interesting to explore if ABIN-1 deficiency improves the necroptosis-based cancer therapy in the CRC xenograft model. Currently, 5fluorouracil (5-FU)-based chemotherapy is still the firstline therapy for CRC. Despite aggressive treatment with 5-FU-based chemotherapy and immunotherapy, the 5-year survival rate for metastatic CRC is only~14% 29 . The main reason is the occurrence of drug resistance, and~50% of metastatic CRC patients show resistance to 5-FU-based chemotherapy 30 . Triggering necroptosis has been proposed as an alternative method of circumventing this problem.
In this study, we found that poly(I:C) + TAK1 inhibitor 5Z-7-oxozeaenol (P5) concurrently induces RDA and necroptosis in Abin-1 −/− mouse embryonic fibroblasts (MEFs), but not in wild-type MEFs. Upon P5 stimulation, cells die by necroptosis in the early stage and die by RDA in the late stage. We further explored if ABIN-1 deficiency sensitizes CRC cells to necroptosis-based cancer therapy. We found that deficiency of ABIN-1 sensitizes CRC cells to both TNF-induced necroptosis and poly(I:C)-induced necroptosis in vitro and in vivo. Two independent xenograft experiments using HT-29 or COLO205 cells show that phase I/II cIAP inhibitor birinapant + clinical caspase inhibitor IDN-6556 (BI) or poly(I:C) + 5Z-7-oxozeaenol +IDN-6556 (P5I) remarkably inhibits tumor growth via necroptosis activation. For poly(I:C)-induced cell death, the sensitizing effect of ABIN-1 deficiency on cell death may be attributed to increased expression of TLR3. Furthermore, for TNF-induced necroptosis, sensitization may be due to increases in TNF-induced RIPK1 polyubiquitination by reducing the recruitment of ubiquitinediting enzyme A20 to the TNFR1 signaling complex and increase in TNF secretion in CRC cells upon pronecroptosis stimulation.
Cell lines, cell culture, siRNA, and lentivirus shRNA CRC cell lines HT-29, COLO205, Caco-2, and HCT116 were from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). COLO205 was cultured in RPMI-1640 supplemented with 10% FBS, penicillin, and streptomycin. HT-29, HCT116, and Caco-2 were cultured in DMEM supplemented with 10% FBS, penicillin, and streptomycin. All cell lines were authenticated by quantitative PCR (qPCR) or western blot assay and tested for mycoplasma contamination.

Cell death assay
Cell death was determined using ToxiLight Nondestructive Cytotoxicity BioAssay Kit (Lonza, Cat# LT07-217). All experiments were conducted on 96-well plates using three biological replicates. Data were collected using Infinite F200 PRO Microplate Reader (Tecan, Swiss).

qPCR analysis and primers
The total RNA was extracted using RNAiso Plus (Trizol) (Takara, Cat# 9108). Reverse transcription was performed with PrimeScript™ RT Reagent Kit with gDNA Eraser

Xenograft experiment
HT-29 or COLO205 cells were transduced with lentivirus-based control shRNA (NC shRNA) or Abin-1 shRNA and screened with puromycin for 5 days to establish stable knockdown pool cells. NC shRNA cells and Abin-1 shRNA cells (5 × 10 6 ) were subcutaneously injected into the left and right flank of nude mice (BALB/c nude mice, 6 weeks of age, from Experimental Animal Center of Yangzhou University, China), respectively. When tumors grew to 100 mm 3 , mice were randomly divided into different groups (2-5 mice each group) and given an intraperitoneal injection with indicated drugs every 3 days. Tumor volumes were measured every 2 or 3 days, and mice were sacrificed 14-18 days after the first drug injection, and tumors were isolated for imaging, weighing, western blot, and immunohistochemical staining. Birinapant, 2.5 mg/kg; IDN-6556, 1.25 mg/kg; poly(I: C), 5 mg/kg; 5Z-7-oxozeaenol, 0.6 mg/kg. Birinapant and IDN-6556 were dissolved in 15% captisol. All animals were housed in microisolator cages, with autoclaved food and bedding to minimize exposure to viral and microbial pathogens, and all procedures were approved by the Institutional Animal Care and Use Committee.

H&E staining
Collected tumor tissues were fixed in 4% PFA and processed for paraffin embedding. The 6-μm-thick histological sections were stained with hematoxylin and eosin by the Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). Images were taken with Olympus BX53 microscope and analyzed using MetaMorph image acquisition software.

Statistics
Data were expressed as means ± standard error of the mean (S.E.M.) and analyzed by two-tailed t tests. Differences were considered statistically significant if *P < 0.05, **P < 0.01, or ***P < 0.001. The experiments were done at least three times in triplicate unless otherwise stated.

ABIN-1 deficiency sensitizes colorectal cancer cells to TNF + birinapant + zVAD/IDN-6656-and TNF + 5-fluorouracil + zVAD/IDN-6556-induced necroptosis
Next, we explored if ABIN-1 deficiency contributes to TNF-triggered necroptosis in CRC cells. It is known that >80% of cancer cell lines are resistant to necroptosis because of incomplete necroptotic machinery, and RIPK3 and MLKL deficiencies are commonly found in numerous cancer cell lines 22,33 . Therefore, we first screened for CRC cell lines that are sensitive to TNF-triggered necroptosis. We chose four common CRC cell lines, Caco-2, HCT116, HT-29, and COLO205, and treated those cells with necroptosis inducers TNF + birinapant+zVAD (TBZ) or TNF + birinapant+IDN-6556 (TBI) in the presence or absence of necrostatin-1 (Nec-1s). We observed that TBZ or TBI can induce RIPK1 phosphorylation in all of those four cells, which was blocked by RIPK1 kinase inhibitor Nec-1s (Fig. 3a-d); however, TBZ or TBI only induced MLKL phosphorylation in HT-29 and COLO205 cells but not in Caco-2 and HCT116 cells (Fig. 3a-d), indicating Caco-2 and HCT116 are defective in the necroptotic signaling pathway and inappropriate for the necroptosis study. Thus, we mainly focused on HT-29 and COLO205 cells in the following study.
To further explore the reason that BI induces necroptosis in absence of TNF, we investigated whether these chemotherapy drugs induce TNF secretion in CRC cells.
Interestingly, although birinapant alone or IDN-6556 alone did not yield increased levels of intracellular TNF and TNF secretions, a combination of birinapant and IDN-6556 resulted in a considerable increase in levels of intracellular TNF and TNF secretions. ABIN-1 deficiency doubled BI-induced TNF expression and secretion (Fig. 4f, g). Suppression of RIPK1 kinase activity by Nec-1s decreased BI-induced TNFα secretion by 70% (Fig. 4f). Not restricted to BI, FI also induced TNF secretion in CRC cells, and ABIN-1 deficiency further enhanced intracellular and extracellular TNF levels, which can be inhibited by Nec-1s (Fig. 4h, i). These results suggest that increased TNF in ABIN-1-deficient CRC cells may explain the higher sensitivity of ABIN-1 deficient CRC cells to BIor FI-induced necroptosis compared to wild-type.

ABIN-1 deficiency augments TNF-induced necroptosis by regulating A20-mediated RIPK1 deubiquitination
We further investigated other mechanisms by which ABIN-1 deficiency sensitizes CRC cells to TNFα-related necroptosis. Previously, we have revealed that in normal cells, ABIN-1 deficiency promotes RIPK1 K63 polyubiquitination and activation of RIPK1 by reducing the recruitment of ubiquitin-editing enzyme A20 to TNF-RSC upon TNF stimulation 16 . We investigated this mechanism in CRC cells that possess a highly variable genome. We found that RIPK1 ubiquitination in TNF-RSC was greatly increased in ABIN-1-deficient HT-29 cells upon TNFα stimulation. A20 was less recruited to TNF-RSC in ABIN-1-deficient cells compared to control cells (Fig. 4j). These results suggest that CRC cells and normal cells share the same mechanism of necroptosis stimulation.

ABIN-1 deficiency enhances necroptosis of human colorectal cancer xenograft
To further investigate if ABIN-1 deficiency improves necroptosis-based cancer therapy, we established a xenograft model by injecting control shRNA CRC cells and Abin-1 shRNA CRC cells into the left flank and right flank of nude mice, respectively, to evaluate if Abin-1 deficiency could enhance necroptosis of tumor cells in vivo. First, we investigated whether ABIN-1 deficiency improves TNF-induced necroptosis in the HT-29 xenograft model. As shown in Fig. 5a (tumor images) and 5b (tumor volume), BI treatment partially inhibited tumor growth compared with normal saline treatment; however, birinapant alone or IDN-6556 alone had no effect. Notably, ABIN-1 deficiency significantly enhanced BIinduced tumor suppression as evidenced by tumor image, tumor volume (P < 0.05 at days 9, 12, and 14), and tumor weight (Fig. 5a-c). To verify that the BI-induced tumor suppression was from necroptosis, western blot analysis of necroptosis markers phospho-RIPK1 and phospho-MLKL in tumor samples was performed. Western blot results showed that BI induced moderate upregulation of RIPK1 and MLKL phosphorylation in control tumors; however, ABIN-1 knockdown greatly increased BI-induced RIPK1 and MLKL phosphorylation ( Fig. 5d and Supplementary  Fig. S5). Immunohistochemical staining results also supported that ABIN-1 deficiency enhanced RIPK1 phosphorylation in response to BI treatment (Fig. 5e).
Furthermore, we explored if ABIN-1 deficiency improves poly(I:C)-induced necroptotic therapy in the COLO205 xenograft model. As shown in Fig. 6a-c, 4 mg/ kg poly(I:C) or 1.25 mg/kg IDN-6556 did not significantly inhibit tumor growth, and 0.6 mg/kg 5Z-7-oxozeaenol only minimally inhibited tumor growth, but P5I induced robust tumor suppression by 50-60%. ABIN-1 deficiency further enhanced P5I-induced tumor suppression flattened the tumor growth curve and reduced the tumor volume and weight (Fig. 6a-c). Western blot analysis of phospho-RIPK1 and phospho-MLKL also supported that active necroptosis occurred in P5I-treated tumors, and ABIN-1 deficiency further enhanced the necroptosis (Fig. 6d). Taken together, we conclude that suppression of ABIN-1 enhances the therapeutic effect of necroptosisbased cancer therapy and increases the probability of slowing tumor progression.

Discussion
In this study, we found that ABIN-1 is a key suppressor for poly(I:C)-induced RDA and necroptosis in MEFs. In the presence of ABIN-1, P5 is insufficient to induce RDA or necroptosis, while deficiency of ABIN-1 permits cells to undergo RDA and necroptosis. Upon poly(I:C) stimulation, TLR3 is engaged and programmed to recruit a complex composed of TRIF, cIAPs, caspase-8, FADD, and RIPK1 to trigger cell death. ABIN-1 deficiency dramatically increases TLR3 expression and may help to recruit more active RIPK1 to initiate RDA and necroptosis. Usually, apoptosis and necroptosis cannot occur at the same time because caspase-8 can cleave RIPK1 at site Asp325 and inhibit the RIPK1 kinase activation and subsequent necroptosis. The classic RDA inducer TNF + 5Z-7-oxozeaenol cannot induce RDA and necroptosis simultaneously 3 . However, ABIN-1 deficiency breaks through the barrier between apoptosis and necroptosis. Previously, we observed that a classic apoptosis inducer TC can induce RIPK1-independent apoptosis and necroptosis concurrently in Abin-1 −/− MEFs. Here, we further reveal that P5 can induce RDA and necroptosis concurrently in Abin-1 −/− MEFs. Interestingly, P5 induces both caspases activation and MLKL phosphorylation in the early stage, but cells die predominantly in a necroptotic pathway; however, in the late stage, caspasesmediated RDA gradually transforms into the principal form of cell death. A possible explanation is ABIN-1 deficiency may dramatically elevate the RIPK1 kinase activity and promptly recruit RIPK3/MLKL to initiate necroptosis; In the meantime, active RIPK1 can recruit FADD, caspase-8 to mediate RDA, and active capase-8, in turn, cleaves RIPK1 to terminate necroptosis, but this process is slower than necrosome formation. Therefore, we first observed P5-induced necroptosis followed by mixed cell death of RDA and necroptosis and lastly only RDA. Contradictory to the results in MEFs, P5 induces RIPK1 kinase-independent apoptosis in CRC cells, but not a mixed cell death of RDA and necroptosis. Many factors have been reported to play a role in the regulation of HT-29 cells were transduced with lentivirusbased control shRNA (NC shRNA) or Abin-1 shRNA and screened with puromycin for 5 days to establish stable knockdown pool cells. NC shRNA cells and Abin-1 shRNA cells were subcutaneously injected into the left and right flank of BALB/c nude mice, respectively. When tumors grew to 100 mm 3 , mice were divided into four groups (normal saline (NaCl), n = 2; IDN-6556 (I), n = 2; birinapant (B), n = 2; birinapant + IDN-6556 (BI), n = 5). Mice were intraperitoneally injected with indicated drugs every three days. Tumor volumes were measured every 2 or 3 days, and mice were sacrificed 14 days after the first drug injection. Tumors were isolated for imaging, weighing, western blot, and immunohistochemical staining. Birinapant, 2.5 mg/kg and IDN-6556, 1.25 mg/kg. a Tumors images. b Tumor volumes. Volume (mm 3 ) = 0.5 × length (mm) × width 2 (mm 2 ). c Tumor weights (g). d Western blot analysis for necroptosis markers. Each lane was loaded with a randomly selected tumor sample from each group. e Immunohistochemical staining with phospho-RIPK1 of tumor samples in BI group. *P < 0.05, **P < 0.01, or ***P < 0.001.
Necroptosis-based cancer therapy is a promising strategy to conquer chemotherapeutic drug resistance; however, not every cancer is suitable for this strategy. Najafov et al. report that 83% of cancer types do not undergo necroptosis. Specifically, in a screen of 941 cell lines, 780 are resistant to TNF + SM-164+zVAD (TSZ)-induced necroptosis. The loss of RIPK3 expression is the major contributing factor in the escape from necroptosis 33 . However, leukemia and CRC seem to be more sensitive to necroptosis compared with other cancer types, and~50% of leukemia and 35% of intestine cancer cell lines are sensitive to TSZ-induced necroptosis 33 . Therefore, CRC is an appropriate cancer type for necroptosis-based therapy. Metzig et al. reported that 5-FU plus IDN-7314, a caspase inhibitor, achieved a better tumor-suppressive effect compared with 5-FU alone in an HT-29 xenograft model. Using 13 unique CRC patient samples, they found that When tumors grew to 100 mm 3 , mice were divided into five groups (normal saline (NaCl), n = 3; poly(I:C), n = 3; 5Z-7-oxozeaenol (5Z-7), n = 3; IDN-6556, n = 3; poly(I:C) + 5Z-7-oxozeaenol+IDN-6556 (P5I), n = 5), Mice were intraperitoneally injected with indicated drugs every 3 days. Tumor volumes were measured every three days, and mice were sacrificed 18 days after the first drug injection. Tumors were isolated for imaging, weighing, and western blot analysis. Poly(I:C), 5 mg/kg; IDN-6556, 1.25 mg/kg; and 5Z-7-oxozeaenol, 0.6 mg/kg. a Tumors images. b Tumor volumes. Volume (mm 3 ) = 0.5 × length (mm) × width 2 (mm 2 ). c Tumor weights (g). d Western blot analysis for necroptosis markers. Each lane was loaded with a randomly selected tumor sample from each group. *P < 0.05, **P < 0.01, or ***P < 0.001. >50% of CRC samples were more sensitive to pronecroptosis agent 5-FU + zVAD than to pro-apoptosis agent 5-FU 27 . These findings suggest a promising benefit of necroptosis-based cancer therapy in CRC patients.
TNF + cIAPs inhibitors + caspase inhibitors is commonly used to induce necroptosis in vitro. However, this combination may not be applicable in vivo because high dose of TNF may evoke a strong inflammation leading to sepsis and multiple organ dysfunction 37 . In addition, since normal tissue may also respond to necroptosis inducers, more research needs to explore targeted necroptosisbased cancer therapy. Our study is one of a few to show FI and BI can induce TNF auto-secretion in cancer cells. Future clinical implications of our study are the possibility of reducing the number of cancer drugs in a regimen from three to two and offering targeted therapy to specific tumor sites since they have higher TNF levels compared to normal tissue. Notably, the combination of BI may be a promising necroptosis-based cancer therapy regimen because both birinapant, a phase I/II anti-cancer reagent and IDN-6556, an FDA-approved pan-caspase inhibitor have been well-studied for individual efficacy and safety profile, and the clinical use of these together may be warranted. In our xenograft experiment, BI treatment did not result in dramatic bodyweight loss or other side effects suggesting a low toxicity profile in BI-based necroptosis therapy in vivo.
Similar to BI, the pro-necroptosis combination P5I also had a robust necroptosis response and low toxicity in the xenograft model. Poly(I:C) is used in conjunction with chemotherapeutic drugs (e.g., cycloheximide, cIAP inhibitors, and arsenic trioxide) to induce apoptosis and play antitumor effects 38,39 . Poly(I:C) also serves as a cancer vaccine adjuvant mainly via facilitating dendritic cell maturation 40 . Takemura et al. 28 reported that poly(I:C) + zVAD (PZ) can induce necroptosis in CT26 colorectal cells without secondary effects of TNFα or type I IFNs. However, our in vitro data showed that PZ and PI do not induce significant cell death in COLO205 cells. In contrast, P5Z and P5I induced more robust necroptosis (Fig.  2e). E6201, a related analog of 5Z-7-oxozeaenol, is in phase I/II clinical trials showing a good tolerance in the treatment of leukemia and solid tumors 41 . Therefore, we propose that poly(I:C) + E6201 + IDN-6556 may be a promising necroptosis inducer in cancer therapy.
For clinical application in necroptosis-based cancer therapy, there are three potential strategies to suppress ABIN-1: (1) develop small molecule inhibitors to block the interaction of ABIN-1 and A20, as this interaction is important for the recruitment of A20 to TNF-RSC to deubiquitinate RIPK1 and suppress necroptosis 16 ; (2) develop folate-, hyaluronic acid-, or antibody-conjugated targeted nanoparticles to specifically deliver Abin-1 siRNA/miRNA to tumor sites 42 ; and (3) Investigate small molecules capable of suppressing ABIN-1 expression. Using publicly available gene expression datasets with connectivity mapping, a commonly used approach to uncover novel medical indications for existing drugs may accelerate the search for ABIN-1 suppressors for clinical use. According to the COSMIC database, 7.14% CRC samples show ABIN-1 overexpression, while 6.04% CRC samples show ABIN-1 underexpression. Suppression of ABIN-1 may not be necessary in underexpressed CRC tissues, but in tumor tissues with normal or overexpression of ABIN-1, targeting ABIN-1 to enhance necroptosis-based therapy may be beneficial. Therefore, characterization of ABIN-1 expression in all cancer types sensitive to necroptosis is warranted.
In conclusion, ABIN-1 is a multiple cell death suppressor, and ABIN-1 deficiency breaks through the barrier of apoptosis and necroptosis in certain conditions. Moreover, necroptosis-based cancer therapy should be considered an alternative option to treat CRC patients with chemotherapeutic drug resistance. Targeting ABIN-1 will further improve this therapy to suppress CRC progression.