Tivozanib mediated inhibition of c-Kit/SCF signaling on Tregs and MDSCs and reversal of tumor induced immune suppression correlates with survival of HCC patients

Abstract

The immune modulatory effect of tivozanib, a tyrosine kinase inhibitor, and the underlying immune mechanisms impacting survival of HCC patients have not been investigated. Pre-clinical studies have shown that tivozanib reduces Tregs and MDSCs accumulation through inhibition of c-Kit/SCF axis. We rationalized that c-Kit/SCF axis antagonism by tivozanib may reverse tumor-induced immune suppression in HCC patients. The frequency of circulating Tregs, MDSCs, CTLA-4+Tregs, PD-1+T cells, c-Kit+pERK-2+Tregs, and c-Kit+pERK-2+MDSCs were quantified in HCC patients at baseline and two time points during tivozanib treatment. We report for the first time that reduction in Tregs after tivozanib treatment and increased levels of baseline CD4+PD-1+T cells correlated with significant improvement in overall survival (OS) of the patients and these signatures may be potential biomarkers of prognostic significance. This immune modulation resulted from tivozanib-mediated blockade of c-Kit/SCF signaling, impacting ERK2 phosphorylation on Tregs and MDSCs. Low pre-treatment CD4+T cells: Treg ratio and reduction in the frequencies of Foxp3+c-Kit+pERK+Tregs after tivozanib treatment correlated significantly with progression free survival. In a comparative analysis of tivozanib vs sorafenib treatment in HCC patients, we demonstrate that decrease in the baseline numbers or frequencies of Foxp3+Tregs, MDSCs and exhausted T cells was significantly greater following tivozanib treatment. Additionally, greater increase in CD4+T cell: Treg ratio after tivozanib treatment was associated with significant improvement in OS compared to sorafenib treatment, highlighting the greater efficacy of tivozanib. These insights may help identify patients who likely would benefit from c-Kit/SCF antagonism and inform efforts to improve the efficacy of tivozanib in combination with immunotherapy.

Trial registration: ClinicalTrials.gov NCT01835223.

Keywords: HCC; MDSC; Tivozanib; Tregs; c-Kit/SCF; survival.

© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.

Figures

Figure 1.

Reduction in Tregs and dynamics of ERK phosphorylation on c-Kit+Tregs during tivozanib therapy (A) Representative histogram offset showing frequency of CD4+Foxp3+ Tregs measured at pre, 28–35d and 160d of tivozanib treatment. (B) Frequency and (C) absolute numbers of CD4+Foxp3+ Tregs pre vs 28–35d (D) Representative histogram offset showing frequency of Foxp3+c-Kit+ Tregs measured at pre, 28–35d and 160d of tivozanib treatment. (E) Frequency and (F) absolute numbers of Foxp3+c-Kit+ Tregs pre vs 28–35d (G) Representative histogram offset showing frequency of c-Kit+pPERK+ Tregs measured at pre, 28–35d and 160d of tivozanib treatment. (H) Frequency and (I) absolute numbers of c-Kit+pPERK+ Tregs pre vs 28–35d (J) Ratio of CD4+CD127+ T cells to CD4+Foxp3+ T cells (CD4+CD127+ T cells/CD4+Foxp3+ T cells) pre vs 28–35d. (K) Ratio of CD8+CD127+ T cells to CD4+Foxp3+ T cells pre vs 28–35d. Each symbol represents an individual HCC patient. Frequencies of Tregs and T effector cells were calculated based on CD3+CD4+ T/CD3+CD8+ T cell population. **** P < 0.0001, *** P < 0. 001, ** P < 0.01, * P < 0.05, paired t-test, Pre vs 28–35d n = 17.

Figure 2.

Reduction in MDSC and dynamics of ERK phosphorylation on c-Kit+ MDSCs during tivozanib therapy. (A) Representative histogram offset showing frequency of CD11b+CD33+ MDSCs measured at pre, 28–35d and 160d of tivozanib treatment. (B) Frequency and (C) absolute numbers of MDSCs pre vs 28–35. (D) Representative histogram offset showing frequency of c-Kit+ MDSCs measured at pre, 28–35d and 160d of tivozanib treatment. (E) Frequency and (F) absolute numbers of c-Kit+ MDSCs pre vs 28–35. (G) Representative histogram offset showing frequency of c-Kit+pERK+ MDSCs measured at pre, 28–35d and 160d of tivozanib treatment. (H) Frequency and (I) absolute numbers of c-Kit+pERK+ MDSCs pre vs 28–35. Each symbol represents an individual HCC patient. Frequencies of MDSCs were calculated based on CD14−HLA-DR− population **** P < 0.0001, *** P < 0. 001, ** P < 0.01, * P < 0.05, paired t-test, n = 17.

Figure 3.

Reduction in immune checkpoint receptors during and after tivozanib therapy. (A) Representative histogram offset showing frequency of Foxp3+CTLA-4+ Tregs measured at pre, 28–35d and 160d of tivozanib treatment. (B) Frequency and (C) absolute numbers of Foxp3+CTLA-4+ Tregs pre vs 28–35d. (D) Representative histogram offset showing frequency of CD4+PD-1+ T cells measured at pre, 28–35d and 160d of tivozanib treatment. (E) Frequency of CD4+PD-1+ T cells pre vs 28–35d (F) Absolute number of CD4+ PD-1+ T cells pre vs 28–35d (G) Representative histogram offset showing frequency of CD8+PD-1+ T cells measured at pre, 28–35d and 160d of tivozanib treatment. (H) Frequency of CD8+PD-1+ T cells pre vs 28–35d (I) Absolute number of CD8+PD-1+ T cells pre vs 28–35d. Each symbol represents an individual HCC patient. Frequencies of CTLA-4+Tregs and PD-1+ T cells were calculated based on CD3+CD4+ T cell/CD3+CD8+ T cell population and. **** P < 0.0001, *** P < 0. 001, ** P < 0.01, * P < 0.05, paired t-test, n = 17.

Figure 4.

Kaplan-Meier plots showing the predictive immune correlates of responses to tivozanib treatment in HCC patient. Association between immunophenotypic signatures and OS or PFS of patients was calculated as described in methods. (A) OS of the patients and increased frequencies of baseline CD4+PD-1+ T cells (HR = 0.92, 95% CI: 0.9–1.0, P = 0.02). (B) Reduction in the frequencies of CD4+Foxp3+ Tregs (post- pre changes) (median reduction: −2.5, range: −7.6 to −0.8), quantified after tivozanib therapy and OS of the patients (HR = 1.6, 95% CI: 1.0–2.3, P = 0.03). (C) Low baseline ratio of CD4+T effector cells to Foxp3+ Tregs and OS of the patients (HR = 1.2, 95% CI: 1.0–1.5, P = 0.046) as well as (D) Low baseline ratio of CD4+T effector cells to Foxp3+ Tregs and PFS of the patients at 24 weeks (HR = 1.4, 95% CI: 1.0–1.9, P = 0.03). (E). Decrease in the frequencies of Foxp3+c-Kit+pERK+Tregs (post-pre changes) and PFS of the patients at 24 weeks (HR = 1.2, 95% CI: 1.0–1.5, P = 0.03).

Figure 5.

Differential effect of tivozanib vs sorafenib treatment on immune suppressive cell subsets in HCC patients and impact of post-treatment changes in the ratio of CD4+T effector cell: Foxp3+ Tregs on overall survival of HCC patients after tivozanib vs sorafenib treatment. Box plots represent mean/median percentage changes from baseline number or frequency of different immune cell subsets after 28–25 days of sorafenib vs. tivozanib treatment (A) Foxp3+ Tregs (B) Foxp3+CTLA-4+ Tregs (C) CD4+PD-1+ T cells (D) CD8+PD-1+ T cells (E) % CD11b+CD33+ MDSC (F) # CD11b+CD33+ MDSC (G) Kaplan-Meir plots showing the association of greater percentage change or increase in the ratio of CD4+T cells: Tregs from baseline and survival probability of HCC patients after sorafenib or tivozanib treatment. P values are shown inside the respective plots; sorafenib n = 49, tivozanib n = 17.