Translational Strategies for Repotrectinib in Neuroblastoma

Abstract

Limited clinical data are available regarding the utility of multikinase inhibition in neuroblastoma. Repotrectinib (TPX-0005) is a multikinase inhibitor that targets ALK, TRK, JAK2/STAT, and Src/FAK, which have all been implicated in the pathogenesis of neuroblastoma. We evaluated the preclinical activity of repotrectinib monotherapy and in combination with chemotherapy as a potential therapeutic approach for relapsed/refractory neuroblastoma. In vitro sensitivity to repotrectinib, ensartinib, and cytotoxic chemotherapy was evaluated in neuroblastoma cell lines. In vivo antitumor effect of repotrectinib monotherapy, and in combination with chemotherapy, was evaluated using a genotypically diverse cohort of patient-derived xenograft (PDX) models of neuroblastoma. Repotrectinib had comparable cytotoxic activity across cell lines irrespective of ALK mutational status. Combination with chemotherapy demonstrated increased antiproliferative activity across several cell lines. Repotrectinib monotherapy had notable antitumor activity and prolonged event-free survival compared with vehicle and ensartinib in PDX models (P < 0.05). Repotrectinib plus chemotherapy was superior to chemotherapy alone in ALK-mutant and ALK wild-type PDX models. These results demonstrate that repotrectinib has antitumor activity in genotypically diverse neuroblastoma models, and that combination of a multikinase inhibitor with chemotherapy may be a promising treatment paradigm for translation to the clinic.

Figure 1:. Repotrectinib Monotherapy In Vivo Therapeutic Drug Studies.

(A) Waterfall plot of repotrectinib monotherapy, ensartinib, and vehicle in PDX models; Solid bars represent ALK WT models and patterned bars represent ALK-mutant models (B) Kaplan-Meier estimates of event-free survival in repotrectinib monotherapy therapeutic studies in the overall cohort (repotrectinib vs vehicle p<0.0001, repotrectinib vs ensartinib =0.02, via log rank) and (C) the ALK-mutant subgroup (repotrectinib vs vehicle p<0.0001, repotrectinib vs ensartinib =0.007, via log rank).

Figure 2:. Repotrectinib Anti-Proliferative Activity in Neuroblastoma Cell Lines as Monotherapy and in Combination with Chemotherapy

(A) Repotrectinib cytotoxicity (IC₅₀) in neuroblastoma cell lines tested stratified by ALK status, MYCN status, and TP53 status; Combination index (CI) of IRI/TMZ/REPO (blue) or IRI/TMZ/ENS (green) in ALK WT (B) or ALK aberrant (C) neuroblastoma cell lines; solid line: median fractional effect; shaded regions: 1.96 x standard error; IRN=irinotecan; TMZ=temozolomide; REPO=repotrectinib, ENS=ensartinib

Figure 3.. Repotrectinib treatment of ALK aberrant cell line and xenograft model shows target engagement of downstream effector pathways.

In vitro: protein levels were assessed by western blot analysis after treatment of cells with the indicated concentrations of repotrectinib: (A) Total ALK and p-ALK in NB-1; (B) Trk and p-Trk in SK-N-DZ and NB-1; (C) using the indicated antibodies to assess multiple downstream signaling pathways in SK-N-DZ and NB-1. (D) In vivo: MSKNBL-40352 (ALK 1275Q, ALK amplified, MYCN amplified) tumor lysates from the three treatment arms (vehicle, ensartinib, and repotrectinib (TPX-0005) were analyzed by western blot using the indicated antibodies.

Figure 4:. Repotrectinib Plus Chemotherapy In Vivo Therapeutic Drug Studies

(A) Waterfall plot of best response after 2 cycles of therapy (MSKNBL-30595, ALK 1174V, MYCN amp, TERT promoter mutation); Tumor volume increase beyond 500% not shown here; (B) Mean tumor volume of MSKNBL-30595 in combination therapy study (n=10 mice per arm); Treatment ongoing throughout study interval. (C) Mean tumor volume of MSKNBL-82180 (ALK WT, MYCN WT, ATRX mutation n=3 mice per arm, exception: IRN/TMZ n=4); IRN=irinotecan; TMZ=temozolomide; REPO=repotrectinib.