Arun S. Singh

Structure-Guided Blockade of CSF1R Kinase in Tenosynovial Giant-Cell Tumor

BACKGROUND Expression of the colony-stimulating factor 1 (CSF1) gene is elevated in most tenosynovial giant-cell tumors. This observation has led to the discovery and clinical development of therapy targeting the CSF1 receptor (CSF1R). METHODS Using x-ray co-crystallography to guide our drug-discovery research, we generated a potent, selective CSF1R inhibitor, PLX3397, that traps the kinase in the autoinhibited conformation. We then conducted a multicenter, phase 1 trial in two parts to analyze this compound. In the first part, we evaluated escalations in the dose of PLX3397 that was administered orally in patients with solid tumors (dose-escalation study). In the second part, we evaluated PLX3397 at the chosen phase 2 dose in an extension cohort of patients with tenosynovial giant-cell tumors (extension study). Pharmacokinetic and tumor responses in the enrolled patients were assessed, and CSF1 in situ hybridization was performed to confirm the mechanism of action of PLX3397 and that the pattern of CSF1 expression was consistent with the pathological features of tenosynovial giant-cell tumor. RESULTS A total of 41 patients were enrolled in the dose-escalation study, and an additional 23 patients were enrolled in the extension study. The chosen phase 2 dose of PLX3397 was 1000 mg per day. In the extension study, 12 patients with tenosynovial giant-cell tumors had a partial response and 7 patients had stable disease. Responses usually occurred within the first 4 months of treatment, and the median duration of response exceeded 8 months. The most common adverse events included fatigue, change in hair color, nausea, dysgeusia, and periorbital edema; adverse events rarely led to discontinuation of treatment. CONCLUSIONS Treatment of tenosynovial giant-cell tumors with PLX3397 resulted in a prolonged regression in tumor volume in most patients. (Funded by Plexxikon; ClinicalTrials.gov number, NCT01004861.).

Tumor-targeting Salmonella typhimurium A1-R in combination with doxorubicin eradicate soft tissue sarcoma in a patient-derived orthotopic xenograft (PDOX) model

A patient with high grade undifferentiated pleomorphic soft-tissue sarcoma from a striated muscle was grown orthotopically in the right biceps femoris muscle of mice to establish a patient-derived orthotopic xenograft (PDOX) model. Twenty PDOX mice were divided into 4 groups: G1, control without treatment; G2, Salmonella typhimurium (S. typhimurium)A1-R administered by intratumoral (i.t.) injection once a week for 4 weeks; G3, doxorubicin (DOX) administered by intraperitoneal (i.p.) injection once a week for 4 weeks; G4, S. typhimurium A1-R (i.t.) administered once a week for 2 weeks followed by i.p. doxorubicin once a week for 2 weeks. On day 25 from the initiation of treatment, tumor volume in G2, G3, and G4 was significantly lower than G1. Mice found without gross tumor included one mouse (20%) in G2; one mouse (20%) in G3; and 3 mice (60%) in G4. Body weight loss did not significantly differ between the 3 treated groups or from the untreated control. Histological examination revealed eradication of tumor only in G4 where mice were treated with S. typhimurium A1-R followed by DOX. Our present study indicates future clinical potential of combining S. typhimurium A1-R with chemotherapy such as DOX for soft tissue sarcoma patients.

High efficacy of tumor-targeting Salmonella typhimurium A1-R on a doxorubicin- and dactolisib-resistant follicular dendritic-cell sarcoma in a patient-derived orthotopic xenograft PDOX nude mouse model

Follicular dendritic-cell sarcoma (FDCS) is a rare and recalcitrant disease. In the present study, a patient-derived orthotopic xenograft (PDOX) mouse model of FDCS was established in the biceps muscle of nude mice. The FDCS PDOX was resistant to both doxorubicin (DOX) and NVP-BEZ235, dactolisib (BEZ) an experimental agent which is a dual pan-phosphoinositide 3-kinase-mammalian target of rapamycin inhibitor. However, in contrast to DOX and BEZ, the FDCS PDOX was sensitive to the tumor-targeting bacterial strain, Salmonella typhimurium A1-R (S. typhimurium A1-R). The combination of S. typhimurium A1-R and either DOX or BEZ did not increase the antitumor efficacy of S. typhimurium A1-R, indicating that DOX and BEZ were not active in this PDOX model. The efficacy of S. typhimurium A1-R in this recalcitrant FDCS gives strong impetus to move bacterial therapy to clinical trials for this disease. The findings of the present study are of particular importance since it demonstrates that S. typhimurium A1-R is effective in a PDOX model of FDCS established from a patient who failed DOX therapy.

Tumor-targeting Salmonella typhimurium A1-R combined with temozolomide regresses malignant melanoma with a BRAF-V600E mutation in a patient-derived orthotopic xenograft (PDOX) model

Melanoma is a recalcitrant disease in need of transformative therapuetics. The present study used a patient-derived orthotopic xenograft (PDOX) nude-mouse model of melanoma with a BRAF-V600E mutation to determine the efficacy of temozolomide (TEM) combined with tumor-targeting Salmonella typhimurium A1-R. A melanoma obtained from the right chest wall of a patient was grown orthotopically in the right chest wall of nude mice to establish a PDOX model. Two weeks after implantation, 40 PDOX nude mice were divided into 4 groups: G1, control without treatment (n = 10); G2, TEM (25 mg/kg, administrated orally daily for 14 consecutive days, n = 10); G3, S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., once a week for 2 weeks, n = 10); G4, TEM combined with S. typhimurium A1-R (25 mg/kg, administrated orally daily for 14 consecutive days and 5 × 107 CFU/100 μl, i.v., once a week for 2 weeks, respectively, n = 10). Tumor sizes were measured with calipers twice a week. On day 14 from initiation of treatment, all treatments significantly inhibited tumor growth compared to untreated control (TEM: p < 0.0001; S. typhimurium A1-R: p < 0.0001; TEM combined with S. typhimurium A1-R: p < 0.0001). TEM combined with S. typhimurium A1-R was significantly more effective than either S. typhimurium A1-R (p = 0.0004) alone or TEM alone (p = 0.0017). TEM combined with S. typhimurium A1-R could regress the melanoma in the PDOX model and has important future clinical potential for melanoma patients.

Nucleoside salvage pathway kinases regulate hematopoiesis by linking nucleotide metabolism with replication stress

Endogenous thymidine plays a critical role in the induction of replication stress in thymocytes.

Effective molecular targeting of CDK4/6 and IGF-1R in a rare FUS-ERG fusion CDKN2A -deletion doxorubicin-resistant Ewing's sarcoma patient-derived orthotopic xenograft (PDOX) nude-mouse model

Ewings sarcoma is a rare and aggressive malignancy. In the present study, tumor from a patient with a Ewings sarcoma with cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) loss and FUS-ERG fusion was implanted in the right chest wall of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. The aim of the present study was to determine efficacy of cyclin-dependent kinase 4/6 (CDK4/6) and insulin-like growth factor-1 receptor (IGF-1R) inhibitors on the Ewings sarcoma PDOX. The PDOX models were randomized into the following groups when tumor volume reached 50 mm3: G1, untreated control; G2, doxorubicin (DOX) (intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, CDK4/6 inhibitor (palbociclib, PD0332991, per oral (p.o.), daily, for 14 days); G4, IGF-1R inhibitor (linsitinib, OSI-906, p.o., daily, for 14 days). Tumor growth was significantly suppressed both in G3 (palbociclib) and in G4 (linsitinib) compared to G1 (untreated control) at all measured time points. In contrast, DOX did not inhibit tumor growth at any time point, which is consistent with the failure of DOX to control tumor growth in the patient. The results of the present study demonstrate the power of the PDOX model to identify effective targeted molecular therapy of a recalcitrant DOX-resistant Ewings sarcoma with specific genetic alterations. The results of this study suggest the potential of PDOX models for individually-tailored, effective targeted therapy for recalcitrant cancer.

Tumor-targeting Salmonella typhimurium A1-R regresses an osteosarcoma in a patient-derived xenograft model resistant to a molecular-targeting drug

Osteosarcoma occurs mostly in children and young adults, who are treated with multiple agents in combination with limb-salvage surgery. However, the overall 5-year survival rate for patients with recurrent or metastatic osteosarcoma is 20-30% which has not improved significantly over 30 years. Refractory patients would benefit from precise individualized therapy. We report here that a patient-derived osteosarcoma growing in a subcutaneous nude-mouse model was regressed by tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R, p<0.001 compared to untreated control). The osteosarcoma was only partially sensitive to the molecular-targeting drug sorafenib, which did not arrest its growth. S. typhimurium A1-R was significantly more effective than sorafenib (P <0.001). S. typhimurium grew in the treated tumors and caused extensive necrosis of the tumor tissue. These data show that S. typhimurium A1-R is powerful therapy for an osteosarcoma patient-derived xenograft model.

Intra-arterial administration of tumor-targeting Salmonella typhimurium A1-R regresses a cisplatin-resistant relapsed osteosarcoma in a patient-derived orthotopic xenograft (PDOX) mouse model.

ABSTRACT Previously, a patient-derived orthotopic xenograft (PDOX) model was established with a lung metastasis from an osteosarcoma patient which developed after adjuvant cisplatinum (CDDP) treatment. In this model, we previously demonstrated the efficacy of trabectedin (TRAB) and temozolomide (TEM) compared with CDDP. In the present report, osteosarcoma tissue was implanted orthotopically in the distal femur of mice which were randomized into the following groups when tumor volume reached approximately 100 mm3; On day 14 after initiation of treatment, all but CDDP significantly inhibited tumor volume growth compared with untreated controls. Control (G1): 793.7 ± 215.0 mm3; CDDP (G2): 588.1 ± 176.9 mm3; Salmonella typhimurium A1-R (S. typhimurium A1-R) intravenous (i.v.) (G3): 269.7 ± 72.7 mm3; S. typhimurium A1-R intra-arterial (i.a.) (G4): 70.2 ± 18.9 mm3 (CDDP: p = 0.056; S. typhimurium A1-R i.v.: p = 0.0001; S. typhimurium A1-R i.a.: p = 0.00003, all vs. untreated controls). i.a. administration of S. typhimurium A1-R was significantly more effective than either CDDP (p = 0.00007), or i.v. administration of S. typhimurium A1-R (p = 0.00007) and significantly regressed the tumor volume compared with day 0 (p = 0.001). The new model of i.a. administration of S. typhimurium A1-R has great promise for the treatment of recalcitrant osteosarcoma.

High Efficacy of Pazopanib on an Undifferentiated Spindle‐cell Sarcoma Resistant to First‐line Therapy Is Identified with a Patient‐derived Orthotopic Xenograft (PDOX) Nude Mouse Model

Undifferentiated spindle‐cell sarcoma (USCS) is a recalcitrant cancer. Our laboratory pioneered the patient‐derived orthotopic xenograft (PDOX) nude mouse model with the technique of surgical orthotopic implantation (SOI). In the present study, we evaluated the efficacy of standard first‐line chemistry of doxorubicin (DOX), gemcitabine (GEM) combined with docetaxel (DOC), compared to pazopanib (PAZ), a multi‐targeting tyrosine‐kinase inhibitor, in an USCS PDOX model. A high‐grade USCS from a striated muscle of the patients was grown orthotopically in the right biceps femoris muscle of nude mice to establish the PDOX model. The PDOX models were randomized into the following groups when tumor volume reached 100 mm3: G1, control without treatment; G2, DOX (3 mg/kg, intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, GEM (100 mg/kg, i.p., weekly, for 2 weeks) combined with DOC (20 mg/kg, i.p., once); G4, PAZ (100 mg/kg, p.o., daily, for 14 days). All treatments except DOX significantly inhibited tumor growth compared to untreated control on day 14 after treatment initiation. Tumor sizes were as fallows: control (G1): 332.0 ± 58.7 mm3; DOX (G2): 316.9 ± 55.9 mm3, P = 0.605; GEM + DOC (G3): 228.9 ± 39.8 mm3, P = 0.001; PAZ (G4): 173.8 ± 23.3 mm3, P < 0.0001. PAZ showed significantly more efficacy compared to other therapies evaluated: DOX (P < 0.0001), GEM + DOC (P = 0.006). There were no animal deaths in any group and body weight of treated mice was not significantly different in each group. The present results demonstrate that the PDOX model of USCS can identify a promising novel agent with significantly greater efficacy than first‐line therapy for this recalcitrant disease. J. Cell. Biochem. 118: 2739–2743, 2017.

Salmonella typhimurium A1-R targeting of a chemotherapy-resistant BRAF-V600E melanoma in a patient-derived orthotopic xenograft (PDOX) model is enhanced in combination with either vemurafenib or temozolomide

ABSTRACT A metastatic melanoma obtained from the right chest wall of a patient was previously established orthotopically in the right chest wall of nude mice as a patient-derived orthotopic xenograft (PDOX) model. We previously showed that the combination of tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) and chemotherapy was highly effective against the melanoma PDOX. In the present study, we investigated the mechanism of the high efficacy of this combination. Two weeks after implantation, 40 PDOX mouse models were randomized into 4 groups of 10 mice each: untreated control (n = 10); treated with S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., once a week for 2 weeks, n = 10); treated with temozolomide (TEM) (25 mg/kg, p.o. for 14 consecutive days) combined with S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., once a week for 2 weeks, n = 10); treated with vemurafenib (VEM) (30 mg/kg, p.o., for 14 consecutive days) combined with S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., once a week for 2 weeks) (n = 10). On day 14 from initiation, all treatments significantly inhibited tumor growth compared with untreated control (S. typhimurium A1-R: p < 0.01; TEM combined with S. typhimurium A1-R: p < 0.01; VEM combined with S. typhimurium A1-R: p < 0.01). Combination therapy with S. typhimurium A1-R was significantly more effective on tumor growth than S. typhimurium A1-R alone (with TEM: p < 0.01; with VEM: p < 0.01). Combination therapy significantly increased S. typhimurium A1-R tumor targeting alone (S. typhimurium A1-R + TEM: p < 0.01, S. typhimurium A1-R + VEM: p < 0.01), relative to S. typhimurium A1-R alone, respectively. In conclusion, chemotherapy drugs promoted targeting of S. typhimurium A1-R of melanoma, thereby enhancing efficacy against the melanoma PDOX.