Utilizing feline oral squamous cell carcinoma patients to develop NQO1-targeted therapy

Abstract

Developing effective therapies for the treatment of advanced head-and-neck squamous cell carcinoma (HNSCC) remains a major challenge, and there is a limited landscape of effective targeted therapies on the horizon. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a 2-electron reductase that is overexpressed in HNSCC and presents as a promising target for the treatment of HNSCC. Current NQO1-targeted drugs are hindered by their poor oxidative tolerability in human patients, underscoring a need for better preclinical screening for oxidative toxicities for NQO1-bioactivated small molecules. Herein, we describe our work to include felines and feline oral squamous cell carcinoma (FOSCC) patients in the preclinical assessment process to prioritize lead compounds with increased tolerability and efficacy prior to full human translation. Specifically, our data demonstrate that IB-DNQ, an NQO1-targeted small molecule, is well-tolerated in FOSCC patients and shows promising initial efficacy against FOSCC tumors in proof-of-concept single agent and radiotherapy combination cohorts. Furthermore, FOSCC tumors are amenable to evaluating a variety of target-inducible couplet hypotheses, evidenced herein with modulation of NQO1 levels with palliative radiotherapy. The use of felines and their naturally-occurring tumors provide an intriguing, often underutilized tool for preclinical drug development for NQO1-targeted approaches and has broader applications for the evaluation of other anticancer strategies.

Keywords: Comparative oncology; Feline; NQO1; Preclinical; Targeted therapy.

Fig. 1

Activity of IB-DNQ single agent against FOSCC. (A) Summary of feline patients in the initial IB-DNQ single agent trial and a description of the IB-DNQ dosing protocol. q.o.wk, once every other week; q.d., every day; q.wk., once a week; DSH, domestic short hair; DLH, domestic long hair. (B) Waterfall plot of patient response (RECIST) and NQO1 staining. Dashed lines denote marker for progressive disease (+20%) vs. partial response (−30%). All responses are considered SD (stable disease). (C) Representative IHC and CT images of 2 clinical cases (Patient 1, top, and Patient 3, bottom). IHC images are at 40x magnification. (D−K) Panel of key hematologic and non-hematologic markers tracked for safety and toxicity monitoring. Dotted lines represent clinical ranges associated for each marker. Not all patients had samples for every time point displayed, so n < 5 at some time points.

Fig. 2

Radiation induces leverageable NQO1 expression in FOSCC tumors. (A) In 3 human HNSCC cell lines and one FOSCC cell line, all which have low basal levels of NQO1 protein and enzymatic activity, NQO1 enzymatic activity is significantly increased at 4 h following 8 Gy radiation. Analysis: unpaired t test, P-values: *P < 0.05; **P < 0.01. (B) Summary of patients enrolled for serial biopsy post-radiation and their initial NQO1 IHC score. DSH, Domestic Short Hair. (C) Tracking NQO1 IHC scores pre-RT, 2 h, and 4 h post-RT. NQO1 scores converge to higher levels upon 8 Gy RT exposure. (D) Representative images of a low NQO1 tumor converting to a high NQO1 expressing tumor upon 8 Gy RT exposure. IHC images are at 40X magnification. (E) NQO1 relative intensities increase upon 8 Gy RT, showing that even high NQO1 expressing tumors have increased intensities of NQO1 staining post-RT treatment. Gray dots denote tumors with NQO1 fold change >10. (F) Representative image of an initially high NQO1 (score 2) staining tumor becoming a more intense NQO1 staining tumor (score 3). IHC images are at 40X magnification. (G) Sequence alignment of NQO1 primary sequence across human, feline, and canine. NQO1*2 polymorphism is defined as a P187S variant in human NQO1. (H) Summary of all feline and canine samples collected, which were all null for NQO1*2 polymorphism.

Fig. 3

Efficacy and tolerability of IB-DNQ + RT in FOSCC patients. (A) Tumor responses of patients (n = 18) who received full protocol of RT (8 Gy) then 2 to 4 h later IB-DNQ (1.0 mg/kg I.V.) once a week for 4 cycles. Each patient corresponds to a separate bar and is colored based on their pre-treatment tumor biopsy NQO1 intensity using IHC. Tumor response was measured by percent tumor change compared to pre-treatment CT and post-treatment CT (taken 1 wk after protocol completion), tumor size was determined according to RECIST guidelines. (B) Representative images of pre-treatment NQO1 IHC, CT and post-treatment CT tumor response images. (C−K) Hematological and non-hematological markers tracked during the course of treatment (n = 18). Normal healthy clinical thresholds for each marker is denoted with a gray dotted line, feline cancer patients often have larger deviations outside normal physiological ranges. (L) Determination of oxidative damage associated with treatment by tracking levels of 8-hydroxy-2′–deoxyguanosine (8-OHdG) in the urine of feline patients. High levels are a clinical indication of oxidative damage, but absolute clinical “normal” thresholds are not agreed upon. (M−O) Pre-treatment and post-extended treatment of patient 14. The total treatment protocol for this patient was the standard RT (8 Gy), then IB-DNQ (1.0 mg/kg I.V.) 2-4 h later for 4 cycles, followed by an additional 10 daily treatments of IB-DNQ (1.0 mg/kg, I.V.) Disease was stable during this treatment course (M, N) and no clinically significant changes in hematocrit was observed during these additional IB-DNQ treatments (O). Gray dotted lines denote the standard threshold for hematocrit percentage for a non-tumor bearing, healthy feline. Statistical analysis for all panels: change from baseline was evaluated with an analysis of variance with post-hoc comparison made with Dunnett's multiple comparisons test, P-values: *P < 0.05; **P < 0.01.