Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. RG7787 is a novel low-immunogenic anti-mesothelin recombinant immunotoxin (RIT), engineered to overcome the limitations of SS1P, a RIT now in clinical trials. In vitro activity was evaluated on five established PDAC cell lines (KLM-1, AsPC-1, BxPC-3, Panc 3.014 and PK-1) and on PDAC cells directly established from a patient tumor (GUMC108). RG7787 had subnanomolar IC50s in most cell lines, and was significantly more active than SS1P in GUMC108, KLM-1 and Panc 3.014 cells. GUMC108 was most sensitive, with RG7787 killing >99% of the cells. In a subcutaneous KLM-1 xenograft mouse model, two cycles of 3 × 2.5 mg/kg RG7787 QOD combined with two cycles of 1 × 50 mg/kg paclitaxel induced near-complete responses, with all tumors regressing below 5 mm3 within 30 days after therapy was initiated (>95% decrease) and no significant growth increase for at least another 3 weeks. RG7787 alone gave limited but significant regressions and paclitaxel by itself arrested tumor growth. Quantifying the uptake of Alexa647-labeled RG7787 in tumors showed that the RIT reached only 45% of KLM-1 cells, accounting in part for the limited responses. Paclitaxel did not improve RG7787 uptake, which thus cannot explain the beneficial effect of the combination therapy. In conclusion, RG7787 has high cytotoxic activity on PDAC cell lines as well as on primary patient cells. In vivo, this novel RIT gives durable near-complete tumor responses when combined with paclitaxel. RG7787 merits further evaluation for the treatment of PDAC.

SummaryCancer cells acquire unlimited proliferative capacity by either re-expressing telomerase or inducing alternative lengthening of telomeres (ALT), which relies on telomere recombination. Here, we show that ALT recombination requires coordinate regulation of the SMX and BTR complexes to ensure the appropriate balance of resolution and dissolution activities at recombining telomeres. Critical to this control is SLX4IP, which accumulates at ALT telomeres and interacts with SLX4, XPF, and BLM. Loss of SLX4IP increases ALT-related phenotypes, which is incompatible with cell growth following concomitant loss of SLX4. Inactivation of BLM is sufficient to rescue telomere aggregation and the synthetic growth defect in this context, suggesting that SLX4IP favors SMX-dependent resolution by antagonizing promiscuous BLM activity during ALT recombination. Finally, we show that SLX4IP is inactivated in a subset of ALT-positive osteosarcomas. Collectively, our findings uncover an SLX4IP-dependent regulatory mechanism critical for telomere maintenance in ALT cancer cells.

The Alternative Lengthening of Telomeres (ALT) pathway stimulates telomere elongation and prevents cellular senescence in approximately 60% of osteosarcoma. While the precise mechanism underlying activation of the ALT pathway is unclear, mutations in the chromatin remodeling protein ATRX, histone chaperone DAXX, and the histone variant H3.3 correlate with ALT status. ATRX and DAXX facilitate deposition of the histone variant H3.3 within heterochromatic regions suggesting that loss of ATRX, DAXX, and/or H3.3 lead to defects in the stability of telomeric heterochromatin. Genetic mutations in ATRX, DAXX, and H3.3 have been detected in ALT positive cancers, however, a subset of ALT samples show loss of ATRX or DAXX protein expression or localization without evidence of genetic alterations suggesting additional uncharacterized defects in ATRX/DAXX/H3.3 function. Here, using Next Generation Sequencing we identified a novel gene fusion event between DAXX and the kinesin motor protein, KIFC3, leading to the translation of a chimeric DAXX-KIFC3 fusion protein. Moreover, we demonstrate that the fusion of KIFC3 to DAXX causes defects in DAXX function likely promoting ALT activity. These data highlight a potentially unrecognized mechanism of DAXX inactivation in ALT positive osteosarcoma and provide rationale for thorough and comprehensive analyses of ATRX/DAXX/H3.3 proteins in ALT positive cancers.

Solid tumors present challenges for delivery of protein therapeutics; current methods cannot quantify the functional effects of these agents. RG7787 (anti-mesothelin recombinant immunotoxin) is highly cytotoxic to pancreatic cancer cell lines, but with limited activity in vivo. To investigate this discrepancy, we developed a flow cytometry method to quantify the amount of RG7787 internalized per cell in tumors and used it to analyze tumor responses by determining the number of molecules of RG7787 internalized per cell in vivo and comparing it to that needed to kill cells in vitro. At a maximum tolerated dose of 7.5 mg/kg, tumor cells in vivo internalized a wide range of RG7787 with the average amount equivalent to the amount that induced growth arrest in vitro. However, 20% of cells accumulated 20,300 ITs per cell, sufficient to kill cells in vitro. At 2.5 mg/kg the top 20% of cells internalized enough RG7787 to only induce growth arrest. These data are in agreement with tumor responses; 22% regression following a 7.5 mg/kg dose and growth stabilization following 2.5 mg/kg. Comparing amounts of RIT delivered in vivo and in vitro can explain tumor responses and should facilitate the development of more active immunotoxins and other antibody based agents.