Improved chemotherapy modeling with RAG-based immune deficient mice

Abstract

We have previously characterized an acute myeloid leukemia (AML) chemotherapy model for SCID-based immune deficient mice (NSG and NSGS), consisting of 5 days of cytarabine (AraC) and 3 days of anthracycline (doxorubicin), to simulate the standard 7+3 chemotherapy regimen many AML patients receive. While this model remains tractable, there are several limitations, presumably due to the constitutional Pkrdcscid (SCID, severe combined immune deficiency) mutation which affects DNA repair in all tissues of the mouse. These include the inability to combine preconditioning with subsequent chemotherapy, the inability to repeat chemotherapy cycles, and the increased sensitivity of the host hematopoietic cells to genotoxic stress. Here we attempt to address these drawbacks through the use of alternative strains with RAG-based immune deficiency (NRG and NRGS). We find that RAG-based mice tolerate a busulfan preconditioning regimen in combination with either AML or 4-drug acute lymphoid leukemia (ALL) chemotherapy, expanding the number of samples that can be studied. RAG-based mice also tolerate multiple cycles of therapy, thereby allowing for more aggressive, realistic modeling. Furthermore, standard AML therapy in RAG mice was 3.8-fold more specific for AML cells, relative to SCID mice, demonstrating an improved therapeutic window for genotoxic agents. We conclude that RAG-based mice should be the new standard for preclinical evaluation of therapeutic strategies involving genotoxic agents.

Fig 1. RAG knockout mice tolerate higher AML therapy than SCID-based mice.

A) Naïve male NSGS mice (n = 3–4, 25.6 +/- 1.1g average body weight) with SCID immune deficiency or B) NRGS male mice (n = 3–4, 27.8 +/- 2.3g) with RAG knockout immune deficiency were challenged with various doses (doses in mg/kg) of combined daunorubicin (D) and Ara-C (A) injection for 3 consecutive days. Survival was monitored to determine maximum tolerable doses. Mice were sacrificed when they reached humane endpoints as described in the methods. (C-H) Mice (n = 4–5 per group) were conditioned with busulfan 3 weeks before exposure to 1.2mg/kg daunorubicin and 50mg/kg Ara-C (BU/DA). Survival (C), relative body weight (D), PB WBCs (E), RBCs (F), hemoglobin (G), and platelets (H) were monitored for responses to chemotherapy. For C-H, 8–12 week old female mice were used with starting weights of 25.4 +/- 3.0g (NRGS) and 24.4 +/- 1.9g (NSGS). Asterisks indicate p<0.05. For C-H, asterisks indicate significant differences between the SCID DA and RAG DA groups. CNTL = PBS controls, DA = combined daunorubicin/Ara-C, WBCs = white blood cells, RBCs = red blood cells.

Fig 2. Lack of efficacy with DA in AML-engrafted NRGS.

A) NRGS mice were engrafted with PDX samples generated from a paired de novo/relapse AML case. Busulfan was used to pre-condition mice for de novo engraftment, but not for mice receiving the relapse sample. Mice were treated with 1.2mg/mL daunorubicin and 50mg/kg Ara-C at 3 weeks and BM aspirates were analyzed at day 25. Survival of the mice engrafted with the B) de novo and C) relapse PDX samples was monitored. Asterisk indicates p<0.05 by Mann-Whitney U test. CNTL = PBS controls, DA = combined daunorubicin/Ara-C. Mice were randomly assigned to treatment or control groups.

Fig 3. Comparison of doxorubicin to daunorubicin in AML PDX models.

A) Survival of NRGS mice engrafted with the MA9.3Ras cell line and treated with 0, 1, 2, or 3 cycles of 1.2mg/kg daunorubicin and 50mg/kg Ara-C (0 DA, 1 DA, 2 DA, 3 DA) or B) 1.5mg/kg doxorubicin and 50mg/kg Ara-C. C) A de novo PDX was engrafted into NRGS mice and treatment began 3 weeks after busulfan conditioning and engraftment using HD DA using either 3.0mg/kg doxorubicin or daunorubicin. AML burden was determined from BM aspirates at day 27. The red points indicate mice with undetectable disease and are plotted as 0.001 in order to include them in the log based plot. D) The mice in C were followed for survival. Log rank tests were used for A,B,D. Mann-Whitney U tests were used to determine significance for C. Asterisks indicate p<0.05. CNTL = PBS controls. Mice in A-D were randomly assigned to treatment or control groups.

Fig 4. HD chemotherapy for de novo and relapse AML PDXs.

A) Survival of NRGS mice engrafted with a matched de novo (DN)–relapsed/refractory (R/R) patient sample were treated with the higher dose of 3.0mg/kg daunorubicin and 75 mg/kg Ara-C. B) Survival of NRGS mice engrafted with a PDX sample from a second de novo case and treated with 1.2mg/kg daunorubicin and 50mg/kg AraC (DA-LD) or a higher dose (DA-HD) as in A. C) NRGS mice engrafted with the AE46T cell line were monitored for AML response to HD DA treatment. D) A relapse adult sample was subjected to two rounds of HD DA chemotherapy. BM AML burden and E) survival are shown. Asterisks indicate p<0.05 by log rank test (panel A, B, E), or 2-way ANOVA (panel C, D). Comparisons are treated versus controls. CNTL = PBS controls. Mice were randomly assigned to treatment or control groups.

Fig 5. Modeling 4-drug induction for high risk B-ALL.

A) Weights of NRG (RAG, n = 10) and NSG (SCID, n = 6) mice were treated with a 4-week course of VXPD. B) Weights of mice conditioned with busulfan 3 weeks prior to VXPD (n = 6 per group). C) Survival of mice in A and B. Mice were sacrificed when they reached humane endpoints as described in the methods. D) WBCs, E) RBCs F) hemoglobin, and G) platelets were monitored before, during, and after VXPD. H) PDX samples from a pediatric B-ALL sample were engrafted into NRG mice after busulfan conditioning. BM aspirates were analyzed by flow cytometry to monitor engraftment. I) Survival and J) B-ALL levels at time of sacrifice were determined for these mice. Asterisks indicate p<0.05 by Mann-Whitney U test (panels A-B, D-H, J) or log rank (panel I). CNTL = PBS controls.

Fig 6. Determination of therapeutic window in NSGS and NRGS mice.

A) Latency of MA9.3RAS leukemia induction in NSGS and NRGS mice. B) Day 14 BM cellularity of mice engrafted with MA9.3RAS cells. Mice were treated with 3 days of PBS (CNTL) or DA (1.2mg/kg daunorubicin and 50mg/kg Ara-C) beginning on day 9. Mice were randomly assigned to treatment or control groups. C) Body Weight plotted against age of mice used in panel B. D) Absolute MA9.3RAS cell number in mice presented in panel B. E) The toxicity ratio was calculated for each DA treated mouse. Log rank test was used for panel A. Asterisks indicate p<0.05 by Mann-Whitney U test (panel B, D, and E) or linear regression analysis (panel C).