Plan comparison of prostate stereotactic radiotherapy in spacer implant patients.

In prostate stereotactic body radiation therapy (SBRT), hydrogel spacers are increasingly used. This study aimed to perform a dosimetry comparison of treatment plans using CyberKnife (CK), commonly used for prostate SBRT, Helical TomoTherapy (HT), and TrueBeam (TB) in patients with hydrogel spacer implantations. The data of 20 patients who received hydrogel spacer implantation for prostate SBRT were retrospectively analyzed. The prescription dose was 36.25 Gy in five fractions to 95% of the planning target volume (PTV; D95). The conformity index (CI), gradient index (GI), homogeneity index (HI), and dose-volume histogram (DVH) were analyzed for the three modalities, using the same PTV margins. The monitor unit (MU) and the beam-on-time (BOT) values were subsequently compared. The CI of TB (0.93 ± 0.02) was significantly superior to those of CK (0.82 ± 0.03, p < 0.01) and HT (0.86 ± 0.03, p < 0.01). Similarly, the GI value of TB (3.59 ± 0.12) was significantly better than those of CK (4.31 ± 0.43, p < 0.01) and HT (4.52 ± 0.24, p < 0.01). The median doses to the bladder did not differ between the CK and TB (V18.1 Gy: 16.5% ± 4.5% vs. 15.8% ± 4.4%, p = 1.00), but were significantly higher for HT (V18.1 Gy: 33.2% ± 7.3%, p < 0.01 vs. CK, p < 0.01 vs. TB). The median rectal dose was significantly lower for TB (V18.1 Gy: 5.6% ± 4.5%) than for CK (V18.1 Gy: 11.2% ± 6.7%, p < 0.01) and HT (20.2% ± 8.3%, p < 0.01). TB had the shortest BOT (2.6 min; CK: 17.4 min, HT: 6.9 min). TB could create treatment plans dosimetrically comparable to those of CK when using the same margins, in patients with hydrogel spacers.

Endogenous Endophthalmitis successfully treated with Intravitreal Povidone-iodine injection: a case report.

BACKGROUND: The usefulness of povidone-iodine as an alternative to antimicrobial agents, for endophthalmitis, has recently been documented. We report a case of endogenous endophthalmitis successfully treated with intravitreal injection of povidone-iodine. CASE PRESENTATION: An 88-year-old woman underwent small bowel bypass surgery for postoperative ileus following rectal cancer resection. She developed a fever during total parenteral nutrition and was diagnosed with gram-positive cocci bacteremia of central venous catheter origin. The patient was referred to our department with chief complaints of ocular pain, hyperemia and decreased vision in the right eye, which had manifested during the febrile period. The initial examination revealed the visual acuity in her right eye to be finger counting and that in her left eye 0.2. The right eye showed a severe inflammatory reaction in the anterior chamber, fibrin deposition, and hypopyon. The fundus was difficult to visualize. Endogenous endophthalmitis due to bacteria was diagnosed. Surgical treatment was judged to be difficult based on the patient's poor general condition and mental status, and intravitreal injection of 0.1 ml of 1.25% povidone-iodine was performed on the same day. The inflammation rapidly diminished, and the hypopyon had disappeared 4 days after treatment. The fundus became visible 7 days after treatment and there was no recurrence of endophthalmitis findings. The visual acuity in her right eye recovered to that in the left eye (0.2). CONCLUSION: Intravitreal injection of povidone-iodine is potentially useful and effective as an alternative treatment of antibiotics for endogenous endophthalmitis patients, especially in whom surgical therapy is difficult.

Difficulty in distinguishing radiation-induced prostate sarcoma from radiation mucositis in a patient with persistent urinary retention and hematuria after prostate cancer radiotherapy.

Urinary retention and hematuria owing to radiation-induced mucositis are occasional late adverse events in patients with prostate cancer. Moreover, radiation-induced secondary malignancies are late adverse events, although they are extremely rare. Herein, we describe a case of radiation-induced secondary malignancy of the prostate that was initially difficult to distinguish from radiation mucositis. A 74-year-old man with prostate cancer underwent brachytherapy and external beam radiotherapy 9 years ago. Twenty-eight months after irradiation, he presented with urinary retention and hematuria owing to radiation mucositis and underwent transurethral resection of the prostate. At 89 months after irradiation, the patient again showed urinary retention and hematuria. The cause of urinary retention and hematuria could not be identified on cystoscopy. Despite receiving medications, the patient's symptoms did not improve. Therefore, transurethral fulguration was performed, and prostate biopsy revealed spindle cell sarcoma. A diagnosis of radiation-induced undifferentiated pleomorphic/spindle cell sarcoma was made, and the patient underwent total cystectomy and construction of the ileal conduit. Two weeks after the surgery, computed tomography revealed peritoneal dissemination. The patient died 5 weeks after the surgery. The case findings indicate that clinicians should consider the possibility of radiation-induced secondary malignancy; moreover, thorough pathological examination of the prostate with CT and MRI is important to distinguish RISM from radiation mucositis even if no tumors are found on cystoscopy.

Anterior Lens Capsule Transplantation for Acquired Optic Disc Pit Maculopathy.

A patient with acquired optic disc pit (ODP) maculopathy underwent vitrectomy with anterior capsule transplantation to the ODP and gas tamponade. Structural changes were evaluated by enhanced depth imaging optical coherence (OCT) tomography. During vitrectomy, the eye was confirmed to have preexisting posterior vitreous detachment. Postoperative OCT showed complete closure of the optic pit resulting in rapid absorption of subretinal fluid. The authors' observations suggest that the anterior capsule is a useful material for achieving optic pit closure. To the authors' knowledge, this is the first report describing application of the anterior capsule to the treatment of ODP maculopathy. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:649-652.].

Prospective study of vitrectomy for epiretinal membranes in patients with good best-corrected visual acuity.

BACKGROUND: To evaluate the efficacy of epiretinal membrane removal in patients with good best-corrected visual acuity (BCVA) for improving visual function and quality of life (QOL). METHODS: This prospective case study compared 37 subjects with preoperative BCVA ≦ 0.046 logMAR (Good group) to 35 patients with 0.10-0.52 logMAR (Moderate group) at 3 and 6 months. Linear mixed-effect models were used for statistical analysis. The primary outcome was the horizontal metamorphopsia score (MH) at 6 months postoperatively (post-6 M), while secondary outcomes were postoperative BCVA, vertical metamorphopsia score (MV), aniseikonia, stereopsis and central foveal thickness. In the Good group, QOL was assessed using the National Eye Institute Visual Functioning Questionnaire-25 (NEI VFQ-25) at 6 and 12 months. RESULTS: MH was significantly improved at post-3 M and post-6 M in the both groups but there were no significant differences between the two groups. MV showed no improvement at the final observation in either group. LogMAR BCVA was significantly improved at post-6 M in the Good group, which had significantly better vision than the Moderate group. Preoperative vertical and horizontal aniseikonia scores remained unchanged in the Good group at post-6 M but worsened in the Moderate group. The NEI VFQ-25 score improved in the Good group, reflecting improved general health, general vision, and mental health. CONCLUSIONS: Early epiretinal surgery for patients with BCVA ≦ 0.046 logMAR was effective for improvement of HM, BCVA, and QOL and prevented worsening of aniseikonia. TRIAL REGISTRATION: UMIN000021220 . Registered 10 September 2015. UMIN Clinical Trials Registry.

No increase in incidence of post-intravitreal injection endophthalmitis without topical antibiotics: a prospective study.

PURPOSE: In our previous report, intravitreal injection using 0.25% povidone-iodine to irrigate the conjunctival sac together with pre- and post-injection topical antibiotics achieved an incidence of post-injection endophthalmitis significantly lower than other reports. In this study, we examined whether similarly low incidence is achieved without using any topical antibiotics. STUDY DESIGN: Prospective cohort study. METHODS: We evaluated intravitreal injections of anti-vascular endothelial growth factor agents conducted by vitreoretinal specialists at the outpatient injection room of a single university hospital. This study had two protocols. First stage: We performed more than 3000 injections with pre-injection but without post-injection topical antibiotics. Final stage: After confirming no case of endophthalmitis in the first stage, we performed more than 12,500 injections without either pre- or post-injection topical antibiotics. In both protocols, we used 0.25% povidone-iodine to sterilize the conjunctival sac both before and after injection. RESULTS: First stage was performed between April 2015 and January 2016. No case of suspected or proven infectious endophthalmitis occurred in 6039 injections [95% confidence interval (CI) 0-0.000497%]. Final stage was performed between February 2016 and November 2017. No case of suspected or proven infectious endophthalmitis occurred in 12,523 injections (95% CI 0-0.00024%). This result was comparable to our previous study using both pre- and post-injection topical antibiotics (0/15,144 injections, 95% CI 0-0.000198%). CONCLUSION: Using conjunctival sac irrigation with 0.25% povidone-iodine before and after intravitreal injection, the incidence of endophthalmitis remains low even when the use of pre- or post-injection topical antibiotics is discontinued.

Development of a Potent Protein Degrader against Oncogenic BCR-ABL Protein.

Chromosomal translocation occurs in some cancer cells, resulting in the expression of aberrant oncogenic fusion proteins that include BCR-ABL in chronic myelogenous leukemia (CML). Inhibitors of ABL tyrosine kinase, such as imatinib and dasatinib, exhibit remarkable therapeutic effects, although emergence of drug resistance hampers the therapy during long-term treatment. An alternative approach to treat CML is to downregulate expression of the BCR-ABL protein. Recently, we have devised a protein knockdown system by hybrid molecules named Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER). This system is designed to induce IAP-mediated ubiquitylation and proteasomal degradation of target proteins. In this review, we describe the development of SNIPER against BCR-ABL, and discuss the features and prospect for treatment of CML.

Acquired Resistance to Shiga Toxin-Induced Apoptosis by Loss of CD77 Expression in Human Myelogenous Leukemia Cell Line, THP-1.

Shiga toxin (Stx) is a main virulence factor of Enterohemorrhagic Escherichia coli (EHEC) that causes diarrhea and hemorrhagic colitis and occasionally fatal systemic complications. Stx induces rapid apoptotic cell death in some cells, such as human myelogenous leukemia THP-1 cells expressing CD77, a receptor for Stx internalization, and the induction of apoptotic cell death is thought to be crucial for the fatal systemic complications. Therefore, in order to suppress the fatal toxicity, it is important to understand the mechanism how cells can escape from apoptotic cell death in the presence of Stx. In this study, we isolated resistant clones to Stx-induced apoptosis from highly sensitive THP-1 cells by continuous exposure with lethal dose of Stx. All of the ten resistant clones lost the expression of CD77 as a consequence of the reduction in CD77 synthase mRNA expression. These results suggest that downregulation of CD77 or CD77 synthase expression could be a novel approach to suppress the fatal toxicity of Stx in EHEC infected patient.

Pharmacological difference between degrader and inhibitor against oncogenic BCR-ABL kinase.

Chronic myelogenous leukemia (CML) is characterized by the oncogenic fusion protein, BCR-ABL protein kinase, against which clinically useful inhibitors have been developed. An alternative approach to treat CML is to degrade the BCR-ABL protein. Recently, potent degraders against BCR-ABL have been developed by conjugating dasatinib to ligands for E3 ubiquitin ligases. Since the degraders contain the dasatinib moiety, they also inhibit BCR-ABL kinase activity, which complicates our understanding of the impact of BCR-ABL degradation by degraders in CML growth inhibition. To address this issue, we chose DAS-IAP, as a potent BCR-ABL degrader, and developed a structurally related inactive degrader, DAS-meIAP, which inhibits kinase activity but does not degrade the BCR-ABL protein. DAS-IAP showed slightly weaker activity than DAS-meIAP in inhibiting cell growth when CML cells were treated for 48 h. However, DAS-IAP showed sustained growth inhibition even when the drug was removed after short-term treatment, whereas CML cell growth rapidly resumed following removal of DAS-meIAP and dasatinib. Consistently, suppression of BCR-ABL levels and downstream kinase signaling were maintained after DAS-IAP removal, whereas kinase signaling rapidly recovered following removal of DAS-meIAP and dasatinib. These results indicate that BCR-ABL degrader shows more sustained inhibition of CML cell growth than ABL kinase inhibitor.

Derivatization of inhibitor of apoptosis protein (IAP) ligands yields improved inducers of estrogen receptor α degradation.

Aberrant expression of proteins often underlies many diseases, including cancer. A recently developed approach in drug development is small molecule-mediated, selective degradation of dysregulated proteins. We have devised a protein-knockdown system that utilizes chimeric molecules termed specific and nongenetic IAP-dependent protein erasers (SNIPERs) to induce ubiquitylation and proteasomal degradation of various target proteins. SNIPER(ER)-87 consists of an inhibitor of apoptosis protein (IAP) ligand LCL161 derivative that is conjugated to the estrogen receptor α (ERα) ligand 4-hydroxytamoxifen by a PEG linker, and we have previously reported that this SNIPER efficiently degrades the ERα protein. Here, we report that derivatization of the IAP ligand module yields SNIPER(ER)s with superior protein-knockdown activity. These improved SNIPER(ER)s exhibited higher binding affinities to IAPs and induced more potent degradation of ERα than does SNIPER(ER)-87. Further, they induced simultaneous degradation of cellular inhibitor of apoptosis protein 1 (cIAP1) and delayed degradation of X-linked IAP (XIAP). Notably, these reengineered SNIPER(ER)s efficiently induced apoptosis in MCF-7 human breast cancer cells that require IAPs for continued cellular survival. We found that one of these molecules, SNIPER(ER)-110, inhibits the growth of MCF-7 tumor xenografts in mice more potently than the previously characterized SNIPER(ER)-87. Mechanistic analysis revealed that our novel SNIPER(ER)s preferentially recruit XIAP, rather than cIAP1, to degrade ERα. Our results suggest that derivatized IAP ligands could facilitate further development of SNIPERs with potent protein-knockdown and cytocidal activities against cancer cells requiring IAPs for survival.

Development of a Small Hybrid Molecule That Mediates Degradation of His-Tag Fused Proteins.

In recent years, the induction of target-protein degradation via the ubiquitin-proteasome system (UPS) mediated by small molecules has attracted attention, and this approach has applications in pharmaceutical development. However, this technique requires a ligand for the target protein that can be incorporated into tailor-made molecules, and there are many proteins for which such ligands have not been found. In this study, we developed a protein-knockdown method that recognizes a His-tag fused to a protein of interest. This strategy theoretically allows comprehensive targeting of proteins of interest by a particular molecule recognizing the tag. As expected, our hybrid molecule 10 [SNIPER(CH6)] efficiently degraded His-tagged CRABP-II and Smad2 in cells. This system provides an easy method to determine the susceptibility of proteins of interest to UPS-mediated degradation. Furthermore, we hope that this method will become an efficient tool to analyze the function of the UPS.

Development of Protein Degradation Inducers of Androgen Receptor by Conjugation of Androgen Receptor Ligands and Inhibitor of Apoptosis Protein Ligands.

Targeted protein degradation using small molecules is a novel strategy for drug development. We have developed hybrid molecules named specific and nongenetic inhibitor of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) that recruit IAP ubiquitin ligases to degrade target proteins. Here, we show novel SNIPERs capable of inducing proteasomal degradation of the androgen receptor (AR). Through derivatization of the SNIPER(AR) molecule at the AR ligand and IAP ligand and linker, we developed 42a (SNIPER(AR)-51), which shows effective protein knockdown activity against AR. Consistent with the degradation of the AR protein, 42a inhibits AR-mediated gene expression and proliferation of androgen-dependent prostate cancer cells. In addition, 42a efficiently induces caspase activation and apoptosis in prostate cancer cells, which was not observed in the cells treated with AR antagonists. These results suggest that SNIPER(AR)s could be leads for an anticancer drug against prostate cancers that exhibit AR-dependent proliferation.

Targeting the Allosteric Site of Oncoprotein BCR-ABL as an Alternative Strategy for Effective Target Protein Degradation.

Protein degradation technology based on hybrid small molecules is an emerging drug modality that has significant potential in drug discovery and as a unique method of post-translational protein knockdown in the field of chemical biology. Here, we report the first example of a novel and potent protein degradation inducer that binds to an allosteric site of the oncogenic BCR-ABL protein. BCR-ABL allosteric ligands were incorporated into the SNIPER (Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers) platform, and a series of in vitro biological assays of binding affinity, target protein modulation, signal transduction, and growth inhibition were carried out. One of the designed compounds, 6 (SNIPER(ABL)-062), showed desirable binding affinities against ABL1, cIAP1/2, and XIAP and consequently caused potent BCR-ABL degradation.

Wet laboratory training using porcine eyes with eyelids.

OBJECTIVE: This study aimed to evaluate the usefulness of a new wet laboratory (wet lab) system using porcine eyes with eyelids. DESIGN: Teaching device trial. PARTICIPANTS: Porcine eyes with orbital tissues and eyelids. METHODS: Twenty porcine eyes with orbital tissues and eyelids were enucleated from pigs butchered at age 6 months. These eyes were positioned in the eye sockets of a model head and stabilized with a pin. Eye draping, dressing with tape, and speculum placement were conducted. The vertical and horizontal widths of the palpebra under the speculum setting were compared with those of 55 patients who underwent cataract surgery. The rotation and torsion of the porcine eye in the new wet lab system were also compared with those of a conventional wet lab system. For comparison with actual cataract surgery, 5 ophthalmologists, including residents, were asked to respond to a questionnaire survey. RESULTS: The horizontal widths of the palpebra under the speculum setting were 27.5 ± 3.1 mm in porcine eyes and 28.6 ± 5.1 mm in human eyes, and the vertical widths were 16.9 ± 1.3 mm and 16.1 ± 1.5 mm (p = 0.53, 0.05). The amounts of rotation and torsion were significantly greater with the new wet lab system. Ophthalmologists evaluated the new wet lab system as being more realistic than the conventional system, in terms of both natural eye movement and restriction of the surgical field by the eyelid and the speculum. CONCLUSIONS: Wet lab training using porcine eyes with eyelids is more practical than older systems as it reproduces an ocular surgical field very similar to that of humans.

Prevalence of hypothyroidism among patients with breast cancer treated with radiation to the supraclavicular field: a single-centre survey.

PURPOSE: We investigated the prevalence of hypothyroidism (HT) in patients with breast cancer who received radiation therapy to the supraclavicular (SC) field to evaluate the effect of radiation on thyroid. METHODS: Between April 2007 and May 2016, consecutive patients with invasive breast cancer who received SC radiation were recruited. Thyroid-stimulating hormone (TSH) and free thyroxine (fT4) were measured between April and August 2016. On the basis of the radiation-planning CT images, thyroid volume was calculated and dose-volume parameters were estimated. The endpoints were the prevalence of HT as determined by high levels of TSH and low levels of fT4 in serum, and the prevalence of subclinical HT, determined by high-serum TSH and normal fT4. RESULTS: Among the 68 consecutive patients, 26 were excluded from evaluation (10 patients died, 6 had a history of previous thyroid disease and 10 were lost to follow-up). One (2.4%) and six (14.3%) of these patients had HT and subclinical HT, respectively, with a mean TSH level of 8.27 µU/mL. By univariate analysis, a predictive factor of HT and subclinical HT was a thyroid volume <8 cm3 (OR 6.44, 95% CI 1.14 to 36.6; p=0.043). Multivariate analysis also showed an association between thyroid volume <8 cm3 and HT or subclinical HT (OR 18.48, 95% CI 1.48 to 230.86; p=0.024). CONCLUSIONS: The prevalence of HT in patients with breast cancer studied was relatively low. Although thyroid volume appeared to be a predictive marker of HT in this cohort, further prospective evaluation is needed.

Development of protein degradation inducers of oncogenic BCR-ABL protein by conjugation of ABL kinase inhibitors and IAP ligands.

Chromosomal translocation occurs in some cancer cells, which results in the expression of aberrant oncogenic fusion proteins that include BCR-ABL in chronic myelogenous leukemia (CML). Inhibitors of ABL tyrosine kinase, such as imatinib and dasatinib, exhibit remarkable therapeutic effects, although emergence of drug resistance hampers the therapy during long-term treatment. An alternative approach to treat CML is to downregulate the BCR-ABL protein. We have devised a protein knockdown system by hybrid molecules named Specific and Non-genetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER), which is designed to induce IAP-mediated ubiquitylation and proteasomal degradation of target proteins, and a couple of SNIPER(ABL) against BCR-ABL protein have been developed recently. In this study, we tested various combinations of ABL inhibitors and IAP ligands, and the linker was optimized for protein knockdown activity of SNIPER(ABL). The resulting SNIPER(ABL)-39, in which dasatinib is conjugated to an IAP ligand LCL161 derivative by polyethylene glycol (PEG) × 3 linker, shows a potent activity to degrade the BCR-ABL protein. Mechanistic analysis suggested that both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP) play a role in the degradation of BCR-ABL protein. Consistent with the degradation of BCR-ABL protein, the SNIPER(ABL)-39 inhibited the phosphorylation of signal transducer and activator of transcription 5 (STAT5) and Crk like proto-oncogene (CrkL), and suppressed the growth of BCR-ABL-positive CML cells. These results suggest that SNIPER(ABL)-39 could be a candidate for a degradation-based novel anti-cancer drug against BCR-ABL-positive CML.

Modifying the planning target volume to optimize the dose distribution in dynamic conformal arc therapy for large metastatic brain tumors.

PURPOSE: When treating large metastatic brain tumors with stereotactic radiotherapy (SRT), high dose conformity to target is difficult to achieve. Employing a modified planning target volume (mPTV) instead of the original PTV may be one way to improve the dose distribution in linear accelerator-based SRT using a dynamic conformal technique. In this study, we quantitatively analyzed the impact of a mPTV on dose distribution. MATERIALS AND METHODS: Twenty-four tumors with a maximum diameter of >2 cm were collected. For each tumor, two plans were created: one used a mPTV and the other did not. The mPTV was produced by shrinking or enlarging the original PTV according to the dose distribution in the original plan. The dose conformity was evaluated and compared between the plans using a two-sided paired t test. RESULTS: The conformity index defined by the Radiation Therapy Oncology Group was 1.34 ± 0.10 and 1.41 ± 0.13, and Paddick's conformity index was 0.75 ± 0.05 and 0.71 ± 0.06, for the plans with and without a mPTV, respectively. All of these improvements were statistically significant (P < 0.05). CONCLUSION: The use of a mPTV can improve target conformity when planning SRT for large metastatic brain tumors.

Reduced anterior chamber contamination by frequent surface irrigation with diluted iodine solutions during cataract surgery.

PURPOSE: To verify that ocular surface irrigation with 0.025% povidone-iodine (PI) or 0.0025% polyvinyl alcohol-iodine (PAI) during cataract surgery minimizes bacterial contamination of the anterior chamber. METHODS: The study was a prospective, interventional case series. First, the bactericidal effect of PI or PAI against Staphylococcus aureus was evaluated in vitro. Next, in 400 eyes undergoing cataract surgery, the ocular surface was irrigated every 20 seconds during surgery with balanced salt solution (BSS; 200 eyes) or BSS containing 0.025% PI (100 eyes) or 0.0025% PAI (100 eyes). At the completion of surgery, anterior chamber fluid was cultured bacteriologically. Visual acuity (VA) and corneal endothelial cell density were measured before and 7 days after surgery. RESULTS: A marked bactericidal effect was observed when S. aureus was directly exposed for 15 seconds to 0.01% PI or 0.001% PAI diluted in BSS. When the two solutions were stored at room temperature, bactericidal effect did not attenuate after 60 min. The bacterial detection rate at the completion of surgery was significantly reduced in 0.025% PI (0%, 0/100 eyes) or 0.0025% PAI group (0%, 0/100 eyes) compared to BSS group (5%, 10/200 eyes) (p = 0.0340). No differences in postoperative visual acuity and postoperative corneal endothelial cell density were observed between three groups. CONCLUSION: In cataract surgery, irrigation every 20 seconds of the operative field with 0.025% PI or 0.0025% PAI, both of which contain 0.0025% available iodine concentration, achieved a very low bacterial contamination rate in the anterior chamber.

In Vivo Knockdown of Pathogenic Proteins via Specific and Nongenetic Inhibitor of Apoptosis Protein (IAP)-dependent Protein Erasers (SNIPERs).

Many diseases, especially cancers, result from aberrant or overexpression of pathogenic proteins. Specific inhibitors against these proteins have shown remarkable therapeutic effects, but these are limited mainly to enzymes. An alternative approach that may have utility in drug development relies on selective degradation of pathogenic proteins via small chimeric molecules linking an E3 ubiquitin ligase to the targeted protein for proteasomal degradation. To this end, we recently developed a protein knockdown system based on hybrid small molecule SNIPERs (Specific and Nongenetic IAP-dependent Protein Erasers) that recruit inhibitor of the apoptosis protein (IAP) ubiquitin ligases to specifically degrade targeted proteins. Here, we extend our previous study to show a proof of concept of the SNIPER technology in vivo By incorporating a high affinity IAP ligand, we developed a novel SNIPER against estrogen receptor α (ERα), SNIPER(ER)-87, that has a potent protein knockdown activity. The SNIPER(ER) reduced ERα levels in tumor xenografts and suppressed the growth of ERα-positive breast tumors in mice. Mechanistically, it preferentially recruits X-linked IAP (XIAP) rather than cellular IAP1, to degrade ERα via the ubiquitin-proteasome pathway. With this IAP ligand, potent SNIPERs against other pathogenic proteins, BCR-ABL, bromodomain-containing protein 4 (BRD4), and phosphodiesterase-4 (PDE4) could also be developed. These results indicate that forced ubiquitylation by SNIPERs is a useful method to achieve efficient protein knockdown with potential therapeutic activities and could also be applied to study the role of ubiquitylation in many cellular processes.

SNIPER(TACC3) induces cytoplasmic vacuolization and sensitizes cancer cells to Bortezomib.

We previously developed a hybrid small molecule SNIPER (Specific and Nongenetic IAP-dependent Protein ERaser) against transforming acidic coiled-coil-3 (TACC3), SNIPER(TACC3), that induces proteasomal degradation of TACC3 protein. In this study, we found that SNIPER(TACC3) induces cytoplasmic vacuolization derived from endoplasmic reticulum (ER) and paraptosis-like cell death selectively in cancer cells. Mechanistic analysis suggests that accumulation of ubiquitylated protein aggregates that requires X-linked inhibitor of apoptosis protein (XIAP) induces ER stress, which results in ER-stress responses involving X-box binding protein-1 (XBP-1) and ER-derived vacuolization in cancer cells. Importantly, inhibition of proteasome enhanced the SNIPER(TACC3)-induced vacuolization, and the combination treatment of SNIPER(TACC3) and bortezomib exhibited a synergistic anticancer activity in several cancer cell lines. The induction of paraptosis-like cell death in cancer cells by SNIPER(TACC3) could be applied to treat cancer cells resistant to undergo apoptosis by overexpression of XIAP.