Masuyo Miyake

Recombinant methioninase (rMETase) is an effective therapeutic for BRAF-V600E-negative as well as -positive melanoma in patient-derived orthotopic xenograft (PDOX) mouse models

Melanoma is a recalcitrant disease. Melanoma patients with the BRAF-V600E mutation have been treated with the drug vemurafenib (VEM) which targets this mutation. However, we previously showed that VEM is not very effective against a BRAF-V600E melanoma mutant in a patient-derived orthotopic xenograft (PDOX) model. In contrast, we demonstrated that recombinant methioninase (rMETase) which targets the general metabolic defect in cancer of methionine dependence, was effective against the BRAF-V600E mutant melanoma PDOX model. In the present study, we demonstrate that rMETase is effective against a BRAF-V600E-negative melanoma PDOX which we established. Forty BRAF-V600E-negative melanoma PDOX mouse models were randomized into four groups of 10 mice each: untreated control (n = 10); temozolomide (TEM) (25 mg/kg, p.o., 14 consecutive days, n = 10); rMETase (100 units, i.p., 14 consecutive days, n = 10); TEM + rMETase (TEM: 25 mg/kg, p.o., rMETase: 100 units, i.p., 14 consecutive days, n = 10). All treatments inhibited tumor growth compared to untreated control (TEM: p = 0.0003, rMETase: p = 0.0006, TEM/rMETase: p = 0.0002) on day 14 after initiation. Combination therapy of TEM and rMETase was significantly more effective than either mono-therapy (TEM: p = 0.0113, rMETase: p = 0.0173). The present study shows that TEM combined with rMETase is effective for BRAF-V600E-negative melanoma PDOX similar to the BRAF-V600E-positive mutation melanoma. These results suggest rMETase in combination with first-line chemotherapy can be highly effective in both BRAF-V600E-negative as well as BRAF-V600E-positive melanoma and has clinical potential for this recalcitrant disease.

Recombinant methioninase in combination with doxorubicin (DOX) overcomes first-line DOX resistance in a patient-derived orthotopic xenograft nude-mouse model of undifferentiated spindle-cell sarcoma

We have previously established a patient-derived orthotopic xenograft (PDOX) model of undifferentiated spindle cell sarcoma (USCS). Recombinant methioninase (rMETase) has previously demonstrated efficacy in PDOX mouse models of human cancers. In the present study, we determined if rMETase in combination with doxorubicin (DOX) can overcome first-line DOX resistance in a PDOX models of USCS. The USCS PDOX mouse models were randomized into the following groups when tumor volume reached 100u202fmm3: G1, control without treatment; G2, doxorubicin (DOX) (3u202fmg/kg, intraperitoneal [i.p.] injection, weekly, for 2 weeks); G3, rMETase (100 units/mouse, i.p., daily, for 2 weeks); G4, DOX (3u202fmg/kg, i.p., weekly, for 2 weeks) combined with rMETase (100 units/mouse, i.p., daily, for 2 weeks). Tumor size and body weight were measured twice a week. On day 14 after initiation, the USCS PDOX tumor sizes were (G1): 360u202f±u202f85u202fmm3; DOX (G2): 355u202f±u202f111u202fmm3, pu202f=u202f.927; rMETase (G3): 182u202f±u202f57u202fmm3, pxa0=xa0.0003; DOXxa0+xa0rMETase (G4): 134xa0±xa029xa0mm3, pu202f=u202f.00001. These results indicate that rMETase can overcome USCS resistance to DOX, which is first line therapy for this disease. The body weight of treated mice was not significantly different in any group. The present results demonstrate the power of the PDOX model to identify effective therapy for recalcitrant cancer and the potential of rMETase to overcome DOX resistance.

Targeting methionine with oral recombinant methioninase (o-rMETase) arrests a patient-derived orthotopic xenograft (PDOX) model of BRAF-V600E mutant melanoma: implications for chronic clinical cancer therapy and prevention

ABSTRACT The elevated methionine (MET) use by cancer cells is termed MET dependence and may be the only known general metabolic defect in cancer. Targeting MET by recombinant methioninase (rMETase) can arrest the growth of cancer cells in vitro and in vivo. We previously reported that rMETase, administrated by intra-peritoneal injection (ip-rMETase), could inhibit tumor growth in a patient-derived orthotopic xenograft (PDOX) model of a BRAF-V600E mutant melanoma. In the present study, we compared ip-rMETase and oral rMETase (o-rMETase) for efficacy on the melanoma PDOX. Melanoma PDOX nude mice were randomized into four groups of 5 mice each: untreated control; ip-rMETase (100 units, i.p., 14 consecutive days); o-rMETase (100 units, p.o., 14 consecutive days); o-rMETase+ip-rMETase (100 units, p.o.+100 units, i.p., 14 consecutive days). All treatments inhibited tumor growth on day 14 after treatment initiation, compared to untreated control (ip-rMETase, p<0.0001; o-rMETase, p<0.0001; o-rMETase+ip-rMETase, p<0.0001). o-rMETase was significantly more effective than ip-rMETase (p = 0.0086). o-rMETase+ip-rMETase was significantly more effective than either mono-therapy: ip-rMETase, p = 0.0005; or o-rMETase, p = 0.0367. The present study is the first demonstrating that o-rMETase is effective as an anticancer agent. The results of the present study indicate the potential of clinical development of o-rMETase as an agent for chronic cancer therapy and for cancer prevention and possibly for life extension since dietary MET reduction extends life span in many animal models.

Tumor-targeting Salmonella typhimurium A1-R is a highly effective general therapeutic for undifferentiated soft tissue sarcoma patient-derived orthotopic xenograft nude-mouse models

Undifferentiated soft tissue sarcoma (USTS) is a recalcitrant and heterogeneous subgroup of soft tissue sarcoma with high risk of metastasis and recurrence. Due to heterogeneity of USTS, there is no reliably effective first-line therapy. We have generated tumor-targeting Salmonella typhimurium A1-R (S.xa0typhimurium A1-R), which previously showed strong efficacy on single patient-derived orthotopic xenograft (PDOX) models of Ewings sarcoma and follicular dendritic cell sarcoma. In the present study, tumor resected from 4 patients with a biopsy-proven USTS (2 undifferentiated pleomorphic sarcoma [UPS], 1 undifferentiated sarcoma not otherwise specified [NOS] and 1 undifferentiated spindle cell sarcoma [USS]) were grown orthotopically in the biceps femoris muscle of mice to establish PDOX models. One USS model and one UPS model were doxorubicin (DOX) resistant. One UPS and the NOS model were partially sensitive to DOX. DOX is first-line therapy for these diseases. S.xa0typhimurium A1-R arrested tumor growth all 4 models. In addition to arresting tumor growth in each case, S.xa0typhimurium A1-R was significantly more efficacious than DOX in each case, thereby surpassing first-line therapy. These results suggest that S.xa0typhimurium A1-R can be a general therapeutic for USTS and possibly sarcoma in general.

Intra-tumor L-methionine level highly correlates with tumor size in both pancreatic cancer and melanoma patient-derived orthotopic xenograft (PDOX) nude-mouse models

An excessive requirement for methionine (MET) for growth, termed MET dependence, appears to be a general metabolic defect in cancer. We have previously shown that cancer-cell growth can be selectively arrested by MET restriction such as with recombinant methioninase (rMETase). In the present study, we utilized patient-derived orthotopic xenograft (PDOX) nude mouse models with pancreatic cancer or melanoma to determine the relationship between intra-tumor MET level and tumor size. After the tumors grew to 100 mm3, the PDOX nude mice were divided into two groups: untreated control and treated with rMETase (100 units, i.p., 14 consecutive days). On day 14 from initiation of treatment, intra-tumor MET levels were measured and found to highly correlate with tumor volume, both in the pancreatic cancer PDOX (p<0.0001, R2=0.89016) and melanoma PDOX (p<0.0001, R2=0.88114). Tumors with low concentration of MET were smaller. The present results demonstrates that patient tumors are highly dependent on MET for growth and that rMETase effectively lowers tumor MET.

Tumor-targeting Salmonella typhimurium A1-R combined with recombinant methioninase and cisplatinum eradicates an osteosarcoma cisplatinum-resistant lung metastasis in a patient-derived orthotopic xenograft (PDOX) mouse model: decoy, trap and kill chemotherapy moves toward the clinic

ABSTRACT In the present study, a patient-derived orthotopic xenograft (PDOX) model of recurrent cisplatinum (CDDP)-resistant metastatic osteosarcoma was treated with Salmonella typhimurium A1-R (S. typhimurium A1-R), which decoys chemoresistant quiescent cancer cells to cycle, and recombinant methioninase (rMETase), which selectively traps cancer cells in late S/G2, and chemotherapy. The PDOX models were randomized into the following groups 14 days after implantation: G1, control without treatment; G2, CDDP (6 mg/kg, intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, rMETase (100 unit/mouse, i.p., daily, for 2 weeks). G4, S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., weekly, for 2 weeks); G5, S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks); G6, S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks) and CDDP (6 mg/kg, i.p. injection, weekly, for 2 weeks). On day 14 after initiation, all treatments except CDDP alone, significantly inhibited tumor growth compared to untreated control: (CDDP: p = 0.586; rMETase: p = 0.002; S. typhimurium A1-R: p = 0.002; S. typhimurium A1-R combined with rMETase: p = 0.0004; rMETase combined with both S. typhimurium A1-R and CDDP: p = 0.0001). The decoy, trap and kill combination of S. typhimurium A1-R, rMETase and CDDP was the most effective of all therapies and was able to eradicate the metastatic osteosarcoma PDOX.

MEK inhibitor trametinib in combination with gemcitabine regresses a patient-derived orthotopic xenograft (PDOX) pancreatic cancer nude mouse model

Pancreatic cancer is resistant to treatment and needs precision individualized therapy to improve the outcome of this disease. Previously, we demonstrated that trametinib (TRA), a MEK inhibitor, could inhibit a pancreatic cancer patient-derived orthotopic xenograft (PDOX). In the present study, we show that gemcitabine (GEM) in combination with TRA was more effective than TRA alone. We implanted a patient pancreatic cancer orthotopically in the pancreatic tail of nude mice to establish the PDOX model. After seven weeks of tumor growth, we divided 32 pancreatic-cancer PDOX nude mice into 4 groups of eight: untreated control; GEM (once a week for 2 weeks); TRA (14 consecutive days); GEMu202f+u202fTRA (GEM: once a week for 2 weeks, TRA:14 consecutive days). We found that treated mice on day 14 had significantly reduced tumor volume in comparison to untreated control. TRA and the combination of GEMu202f+u202fTRA therapy significantly inhibited tumor development in comparison to GEM alone. However, GEMu202f+u202fTRA inhibited the PDOX tumor growth significantly greater than TRA alone. These results suggest the clinical potential of the combination of TRA and GEM for pancreatic cancer.

Individualized doxorubicin sensitivity testing of undifferentiated soft tissue sarcoma (USTS) in a patient-derived orthotopic xenograft (PDOX) model demonstrates large differences between patients

ABSTRACT Doxorubicin (DOX) is often first-line treatment of undifferentiated/unclassified soft tissue sarcoma (USTS). However, the DOX response rate for USTS patients is low. Individualized precision-medicine technology that could identify DOX responders as well as non-responders would be of high value to cancer patients. In the present study, we established 5 patient-derived orthotopic xenograft (PDOX) nude mouse models from 5 USTS patients and evaluated the efficacy of DOX in each PDOX model. USTSs were grown orthotopically in the right thigh of nude mice to establish the PDOX models. Two weeks after implantation, the mouse models were randomized into two groups of 8 mice each: untreated control; and DOX (3 mg/kg, i.p., once a week for 2 weeks). DOX showed significant growth inhibition in only 2 USTS PDOX models out of 5 (p = 0.0054, p = 0.0055, respectively) on day 14 after initiation. DOX was ineffective in the other 3 PDOX models. However, even in the DOX-sensitive cases, DOX could not regress the PDOX tumors responding to treatment. The present study has important implications since this is the first in vivo study to compare the DOX sensitivity for USTS on multiple patient tumors. We showed that only two of five USTS were responsive to DOX, despite DOX being first line chemotherapy for USTS. The 3 resistant cases should not be treated with DOX clinically, in order to spare the patients unnecessary toxicity. This PDOX model is useful for precise individualized drug sensitivity testing, especially for rare heterogeneous recalcitrant sarcomas such as USTS.

Temozolomide combined with irinotecan regresses a cisplatinum-resistant relapsed osteosarcoma in a patient-derived orthotopic xenograft (PDOX) precision-oncology mouse model

Relapsed osteosarcoma is a recalcitrant tumor. A patients cisplatinum (CDDP)-resistant relapsed osteosarcoma lung metastasis was previously established orthotopically in the distal femur of mice to establish a patient-derived orthotopic xenograft (PDOX) model. In the present study, the PDOX models were randomized into the following groups when tumor volume reached 100 mm3: G1, control without treatment; G2, CDDP (6 mg/kg, intraperitoneal (i.p.) injection, weekly, for 2 weeks); gemcitabine (GEM) (100 mg/kg, i.p., weekly, for 2 weeks) combined with docetaxel (DOC) (20 mg/kg, i.p., once); temozolomide (TEM) (25 mg/kg, p.o., daily, for 2 weeks) combined with irinotecan (IRN) (4 mg/kg i.p., daily for 2 weeks). Tumor size and body weight were measured with calipers and a digital balance twice a week. After 2 weeks, all treatments significantly inhibited tumor growth except CDDP compared to the untreated control: CDDP: p = 0.093; GEM+DOC: p = 0.0002, TEM+IRN: p < 0.0001. TEM combined with IRN was significantly more effective than either CDDP (p = 0.0001) or GEM combined with DOC (p = 0.0003) and significantly regressed the tumor volume compared to day 0 (p = 0.003). Thus the PDOX model precisely identified the combination of TEM-IRN that could regress the CDDP-resistant relapsed metastatic osteosarcoma PDOX.

Doxorubicin-resistant pleomorphic liposarcoma with PDGFRA gene amplification is targeted and regressed by pazopanib in a patient-derived orthotopic xenograft mouse model

Pleomorphic liposarcoma (PLPS) is a heterogeneous resistant group of tumors. Complete surgical resection is the only known way to treat PLPS. PLPS is reristant to both radiation and chemotherapy. Therefore, precise individualized therapy is needed to improve outcome of advanced PLPS patients. In this study, a patient-derived orthotopic xenograft (PDOX) model of a PDGFRA-amplified PLPS was established in the biceps femoris of nude mice by surgical orthotopic implantation (SOI) in order to match the patient. The PLPS PDOX was treated with pazopanib (PAZ) which targets PDGFRA, as well as with temozolomide (TEM) and first-line therapy doxorubicin (DOX). The PLPS PDOX was resistant to DOX and responded very well to PAZ as well as TEM. The tumor volume on treatment day-14 relative to day-1 was as follows: DOX (4.50u202f±u202f2.6, pu202f=u202f0.8087); PAZ (1.29u202f±u202f0.9, pu202f=u202f0.0008 compared to the control, pu202f=u202f0.0167 compared to DOX); TEM (1.07u202f±u202f0.8, pu202f=u202f0.0079 compared to the control, pu202f=u202f0.0079 compared to DOX). There was no significant difference in body weight between any treated group or control. The PAZ- and TEM-treated tumors showed extensive necrosis compared to the DOX-treated and untreated PDOX tumors. The present study showed that PDGFRA amplification could be effectively targeted by PAZ. The PLPS PDOX model also identified the efficacy of TEM which does not target PDGFRA, indicating that the PDOX model can identify effective targeted therapy as well as standard therapy and at the same time, identify ineffective drugs, even if they are first-line.