Inhibition of the NLRP3 inflammasome using MCC950 reduces vincristine-induced adverse effects in an acute lymphoblastic leukemia patient-derived xenograft model

Abstract

Vincristine is one of the most important chemotherapeutic drugs used to treat acute lymphoblastic leukemia (ALL). Unfortunately, vincristine often causes severe adverse effects, including sensory-motor neuropathies, weight loss, and overall decreased well-being, that are difficult to control and that decrease the quality of life and survival of patients. Recent studies demonstrate that sensory-motor adverse effects of vincristine are driven by neuroinflammatory processes, including the activation of the Nod-like receptor 3 (NLRP3) inflammasome. In this study, we aimed to test the effects of MCC950, a specific NLRP3 inhibitor, on the prevention of vincristine-induced adverse effects as well as tumor progression and vincristine efficacy in NOD/SCID/interleukin-2 receptor γ-negative mice patient-derived xenografts of ALL. We demonstrate that co-administration of MCC950 effectively prevented the development of mechanical allodynia, motor impairment, and weight loss and significantly improved the overall well-being of the animals without negatively impacting the in vivo efficacy of vincristine as a single agent or in combination with standard-of-care drugs. These results provide proof of principle that the adverse effects of vincristine chemotherapy can be prevented using NLRP3 inflammasome inhibitors and provide new options for the development of effective treatment strategies.

Figure 1

MCC950 dose‐finding study for the prevention of mechanical allodynia, weight loss, and altered well‐being of mice treated with vincristine. (A) Injection and experimental schedule of vincristine‐induced adverse effects model. Vincristine (V, 0.5 mg/kg, i.p.) was injected once a week for 4 weeks. MCC950 (5–20 mg/kg, i.p.) or saline (blue) were injected 1 h before the first vincristine administration and then daily for 4 weeks. Von Frey and parallel rod floor (PRF) apparatus assays were performed twice a week, and weight measurements and blood analyses were performed once a week as indicated in the schedule for 4 weeks. Animals were observed for an additional week (week 5). (B) Vincristine‐induced mechanical allodynia, measured as the decline of the mechanical paw withdrawal threshold (PWT, g) is alleviated by daily co‐administration of MCC950 (10–20 mg/kg, i.p.). (C–E) MCC950 had no effect on ataxia index (C), lymphocyte counts (D), and weight (E). (F) Animals treated with vincristine together with increasing doses of MCC950 have lower cumulative adverse effects cores compared to animals treated with vincristine only. Statistical significance was determined by repeated measures two‐way ANOVA with Sidak's multiple comparisons test (A–E) or one‐way ANOVA (F); data are shown as mean ± SEM. n ≥ 6/group. #, p < 0.05.

Figure 2

MCC950 may allow for vincristine therapy continuation by alleviating vincristine‐induced sensory neuropathy in NSG mice. (A–C) Mechanical allodynia induced by the treatment with lower doses of vincristine (0.1–0.5 mg/kg, i.p., black dots) is attenuated by daily co‐treatment with MCC950 (15 mg/kg, i.p., blue dots). (D–F) Mechanical allodynia induced by treatment with higher doses of vincristine (0.75–1.25 mg/kg, i.p., black dots) is partially attenuated by daily co‐treatment with MCC950 (15 mg/kg, i.p., blue dots). The gray bar indicates the time frame of daily MCC950 (or saline) administration. The black arrows indicate the time points of vincristine administration. (G) The co‐administration of MCC950 allows for the continuation of the vincristine dose by alleviating sensory neuropathy symptoms. The estimated ED₅₀ of mechanical allodynia caused by vincristine used in combination with MCC950 is estimated 10× higher (0.7 mg/kg, 90% CI 0.6–0.7) compared to the ED₅₀ of mechanical allodynia caused by vincristine alone (0.07 mg/kg, 90% CI 0.06–0.09). The ED₅₀ was calculated using the area under the curve of the experimental values following the various doses of vincristine (A–F) with or without the addition of MCC950. Statistical significance was determined using repeated measures two‐way ANOVA with Sidak's multiple comparisons test. All data are shown as mean ± SEM; n = 6 for all groups. #, p < 0.05. PWT, paw withdrawal threshold.

Figure 3

MCC950 co‐administration alleviates adverse effects induced by high doses of vincristine. (A–C) Motor impairment, measured as an increase in ataxia index using the Parallel Rod Floor apparatus, caused by higher doses of vincristine (0.7–1.25 mg/kg, i.p., black dots) is partially alleviated by co‐administration of MCC950 (15 mg/kg, i.p., blue dots. (D–F) The increase in weight over time of groups treated with higher doses of vincristine (0.75–1.25 mg/kg, i.p.) and co‐treated with MCC950 (15 mg/kg, i.p.) (blue dots) was faster compared to the groups receiving high vincristine doses only (black dots). (G–I) Co‐administration of MCC950 (15 mg/kg, i.p.) with high doses of vincristine (0.75–1.25 mg/kg, i.p.) (blue dots) is not significantly impacting the levels of lymphocytes in plasma compared to vincristine treatment alone (black dots). (J–L) Co‐administration of MCC950 (15 mg/kg, i.p.) with high doses of vincristine (0.75–1.25 mg/kg, i.p.) (blue dots) reduces cumulative scores for appearance and weight loss caused by vincristine (black dots). Statistical significance was determined using repeated measures two‐way ANOVA with Sidak's multiple comparisons test (A–I) or unpaired multiple t‐test (J–L). Group comparison: V (respective dose, 0.75–1.25 mg/kg) + MCC950 compared to V (respective dose, 0.75–1.25 mg/kg) alone. All data are shown as mean ± SEM; n = 6 for all groups. #, p < 0.05.

Figure 4

Increased expression of NLRP3 in pediatric blood cancer types relative to other cancer types and healthy tissue. (A) Leukemia types overexpress NLRP3 compared to other cancer types. (B) The NLRP3 expression levels of the PPTC PDX data set are similar to the NLRP3 expression levels found in St. Jude's data set. (C) Comparison of the NLRP3 expression levels in the PPTC data set to normal tissue NLRP3 expression (GTEx data set). ASPS, alveolar soft part sarcoma; ATRT, atypical teratoid rhabdoid tumor; BCP‐ALL, B‐cell precursor ALL; CNS‐EFT‐CIC, CNS Ewing sarcoma family tumor with CIC Alteration; CNS Embr. NOS, CNS embryonal tumors, not otherwise specified; DIPG, diffuse intrinsic pontine glioma; ETMR, embryonal tumor with multilayered rosettes; ETP‐ALL, Early T‐cell Precursor ALL; iAMP, intrachromosomal amplification of chromosome 21; MLL‐ALL, MLL/KMT2A‐rearranged ALL; MRT, malignant rhabdoid tumors; NOS, not otherwise specified; Ph+ ALL, Philadelphia Chromosome‐positive ALL; Ph‐like‐ALL, Ph‐like ALL; RGA, recurrent genomic abnormalities, which encompasses B‐ALLs with known genomic abnormalities that do not have their own OncoTree subtype classification (https://oncotree.mskcc.org/); RMS, rhabdomyosarcoma; SC, small cell; T‐ALL, T‐cell ALL.

Figure 5

MCC950 co‐administration has no effect on tumor progression or vincristine efficacy in ALL‐19 PDX model. (A) ALL‐19 engraftment and injection schedule. Vincristine (0.1, 0.3, or 0.75 mg/kg, i.p.) was administered once a week for 4 weeks. MCC950 (15 mg/kg, i.p.) was administered 1 h before the first vincristine administration and then daily for 4 weeks. Human CD45⁺ lymphocytes in peripheral blood (PB) were analyzed once a week until the endpoint was reached (huCD45+ cells ≥ 25% in the PB). (B) Overview of ALL‐19 progression (measured as a percentage of huCD45⁺ cells in plasma) over time. Data are shown as single animal data points. (C) Event‐free survival (EFS): there were no significant differences in EFS of groups treated with MCC950 alone (purple dotted line) or vehicle (black line). A higher dose of vincristine 0.3 mg/kg (bright green) or 0.75 mg/kg (red) significantly (p < 0.05) increases the EFS compared to a low dose of vincristine 0.1 mg/kg (blue line). The addition of MCC950 to vincristine 0.3 mg/kg (violet) or 0.75 mg/kg treatment (gray) had no significant negative effect on EFS compared to vincristine 0.3 or 0.75 mg/kg treatment alone. The statistical significance of EFS was determined using ordinary one‐way ANOVA, with Tukey's multiple comparison test. Individual data are shown. n ≥ 6/group. #, p < 0.05.

Figure 6

MCC950 co‐administration has no effect on VXL efficacy and tumor progression in the ALL‐19 PDX model. (A) ALL‐19 engraftment and injection schedule. (B, C) Treatment with vincristine (V), dexamethasone (X), and L‐asparaginase (L) (VXL, red lines) alone or in combination with MCC950 (VXL + MCC950, blue lines) significantly (p < 0.05) increase the event‐free survival (EFS) compared to saline (black lines). The addition of MCC950 to VXL (VXL + MCC950, blue lines) has no significant impact on the EFS compared to VXL alone (p = 0.6702) (VXL, red lines). Statistical significance of EFS was determined using ordinary one‐way ANOVA, with Tukey's multiple comparison test. Individual data are shown. n ≥ 6/group.

Figure 7

MCC950 co‐administration prevents adverse effects induced by combination treatment with vincristine, L‐asparaginase, and dexamethasone (VXL). (A) The combination of vincristine (0.3 mg/kg, i.p.), dexamethasone (5 mg/kg, i.p.), L‐asparaginase (1250 KU/kg, i.p.) (VXL, black dots) causes a significant decrease of paw withdrawal thresholds (PWT) in NSG mice, which was prevented by co‐treatment with MCC950 (15 mg/kg, i.p.) (VXL + MCC950, blue dots). (B) MCC950 co‐administration (blue dots) prevented VXL‐induced decrease in grip strength (Black dots). (C) Motor impairment, measured as an increase in ataxia index, caused by VXL (black dots) was unaffected by MCC950 treatment. (D) Significant differences in weight gain between groups treated with VXL alone (black dots) or co‐treated with MCC950 (blue dots). (E) Co‐administration of MCC950 with VXL (blue dots) did not significantly impact the levels of lymphocytes in plasma compared to VXL treatment alone (black dots). (F) Co‐administration of MCC950 with VXL (blue dots) significantly reduced cumulative adverse effects scores for locomotion and appearance caused by VXL (black dots). The statistical significance was determined using repeated measures two‐way ANOVA with Sidak's multiple comparisons test (A–E) or unpaired multiple t‐tests (F). All data are shown as mean ± SEM; n = 6 for all groups. #, p < 0.05.