Acquired evasive resistance is a major limitation of hepatocellular carcinoma (HCC) treatment with the tyrosine kinase inhibitor (TKI) sorafenib. Recent findings suggest that resistance to sorafenib may have a reversible phenotype. Additionally, loss of responsiveness has been proposed to be due to a gradual decrease in sorafenib plasma levels in patients. Here, the possible mechanisms underlying reversible sorafenib resistance were investigated using a Hep3B-hCG orthotopic human xenograft model of locally advanced HCC. Tissue and plasma sorafenib and metabolite levels, downstream anti-tumor targets and toxicity were assessed during standard and dose-escalated sorafenib treatment. Drug levels were found to decline significantly over time in mice treated with 30 mg/kg sorafenib coinciding with the onset of resistance but a greater magnitude of change was observed in tissues compared to plasma. Skin rash also correlated with drug levels and tended to decrease in severity over time. Drug level changes appeared to be partially tumor-dependent involving induction of tumoral CYP3A4 metabolism, with host pre-treatment alone unable to generate resistance. Escalation from 30 to 60 mg/kg sorafenib improved anti-tumor efficacy but worsened survival due to excessive body weight loss. Microvessel density was inhibited by sorafenib treatment but remained stable over time and dose increase. In conclusion tumor CYP3A4 induction by sorafenib is a novel mechanism to account for variability in systemic drug levels, however declining systemic sorafenib levels may only be a minor resistance mechanism. Escalating the dose may be an effective treatment strategy, provided toxicity can be controlled.

Increasing evidence suggests that inflammatory cytokines play a critical role in tumor initiation and progression. A cancer stem cell (CSC)-like subpopulation in neuroblastoma is known to be marked by expression of the G-CSF receptor (G-CSFR). Here, we report on the mechanistic contributions of the G-CSFR in neuroblastoma CSCs. Specifically, we demonstrate that the receptor ligand G-CSF selectively activates STAT3 within neuroblastoma CSC subpopulations, promoting their expansion in vitro and in vivo. Exogenous G-CSF enhances tumor growth and metastasis in human xenograft and murine neuroblastoma tumor models. In response to G-CSF, STAT3 acts in a feed-forward loop to transcriptionally activate the G-CSFR and sustain neuroblastoma CSCs. Blockade of this G-CSF–STAT3 signaling loop with either anti-G-CSF antibody or STAT3 inhibitor depleted the CSC subpopulation within tumors, driving correlated tumor growth inhibition, decreased metastasis, and increased chemosensitivity. Taken together, our results define G-CSF as a CSC-activating factor in neuroblastoma, suggest a comprehensive reevaluation of the clinical use of G-CSF in these patients to support white blood cell counts, and suggest that direct targeting of the G-CSF–STAT3 signaling represents a novel therapeutic approach for neuroblastoma. Cancer Res; 75(12); 1–14. ©2015 AACR.

Increasing evidence suggests that inflammatory cytokines play a critical role in tumor initiation and progression. A cancer stem cell-like (CSC) subpopulation in neuroblastoma is known to be marked by expression of the G-CSF receptor. Here we report on the mechanistic contributions of the G-CSF receptor in neuroblastoma CSCs. Specifically, we demonstrate that the receptor ligand G-CSF selectively activates STAT3 within neuroblastoma CSC subpopulations, promoting their expansion in vitro and in vivo. Exogenous G-CSF enhances tumor growth and metastasis in human xenograft and murine neuroblastoma tumor models. In response to G-CSF, STAT3 acts in a feed-forward loop to transcriptionally activate the G-CSF receptor and sustain neuroblastoma CSCs. Blockade of this G-CSF/STAT3 signaling loop with either anti-G-CSF antibody or STAT3 inhibitor depleted the CSC subpopulation within tumors, driving correlated tumor growth inhibition, decreased metastasis and increased chemosensitivity. Taken together, our results define G-CSF as a CSC activating factor in neuroblastoma, suggest a comprehensive re-evaluation of the clinical use of G-CSF in these patients to support white blood cell counts, and suggest that direct targeting of the G-CSF/STAT3 signaling represents a novel therapeutic approach for neuroblastoma.

A key challenge in the field of T-cell immunotherapy for cancer is creating a suitable platform for promoting differentiation of effector cells while at the same time enabling self-renewal needed for long-term memory. Although transfer of less differentiated memory T cells increases efficacy through greater expansion and persistence in vivo, the capacity of such cells to sustain effector functions within immunosuppressive tumor microenvironments may still be limiting. We have therefore directly compared the impact of effector versus memory differentiation of therapeutic T cells in tumor-bearing mice by introducing molecular switches that regulate cell fate decisions via mTOR. Ectopic expression of RAS homolog enriched in brain (RHEB) increased mTORC1 signaling, promoted a switch to aerobic glycolysis, and increased expansion of effector T cells. By rapidly infiltrating tumors, RHEB-transduced T cells significantly reduced the emergence of immunoedited escape variants. In contrast, expression of proline-rich Akt substrate of 40 kDa (PRAS40) inhibited mTORC1, promoted quiescence, and blocked tumor infiltration. Fate mapping studies following transient expression of PRAS40 demonstrated that mTORC1low T cells made no contribution to initial tumor control but instead survived to become memory cells proficient in generating recall immunity. Our data support the design of translational strategies for generating heterogeneous T-cell immunity against cancer, with the appropriate balance between promoting effector differentiation and self-renewal. Unlike pharmacologic inhibitors, the genetic approach described here allows for upregulation as well as inhibition of the mTORC1 pathway and is highly selective for the therapeutic T cells without affecting systemic mTORC1 functions. Cancer Res; 75(13); 1–12. ©2015 AACR.

A key challenge in the field of T cell immunotherapy for cancer is creating a suitable platform for promoting differentiation of effector cells while at the same time enabling self-renewal needed for long-term memory. Although transfer of less differentiated memory T cells increases efficacy through greater expansion and persistence in vivo, the capacity of such cells to sustain effector functions within immunosuppressive tumor microenvironments may still be limiting. We have therefore directly compared the impact of effector versus memory differentiation of therapeutic T cells in tumor-bearing mice by introducing molecular switches that regulate cell fate decisions via mTOR. Ectopic expression of RAS homologue enriched in brain (RHEB) increased mTOR complex 1 (mTORC1) signaling, promoted a switch to aerobic glycolysis and increased expansion of effector T cells. By rapidly infiltrating tumors, RHEB-transduced T cells significantly reduced the emergence of immunoedited escape variants. In contrast, expression of proline-rich Akt substrate of 40kDa (PRAS40) inhibited mTORC1, promoted quiescence and blocked tumor infiltration. Fate mapping studies following transient expression of PRAS40 demonstrated that mTORC1low T cells made no contribution to initial tumor control but instead survived to become memory cells proficient in generating recall immunity. Our data support the design of translational strategies for generating heterogeneous T cell immunity against cancer, with the appropriate balance between promoting effector differentiation and self-renewal. Unlike pharmacological inhibitors, the genetic approach described here allows for up-regulation as well as inhibition of the mTORC1 pathway, and is highly selective for the therapeutic T cells without affecting systemic mTORC1 functions.