https://orcid.org/0000-0002-3255-0467
Abstract
Acquired resistance to temozolomide (TMZ) chemotherapy due to DNA mismatch repair (MMR) enzyme deficiency is a barrier to improving outcomes for isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM) patients. KL-50 is a new imidazotetrazine-based therapeutic designed to induce DNA interstrand cross-links, and subsequent double-stranded breaks, in an MMR-independent manner in cells with O-6-methylguanine-DNA methyltransferase (MGMT) deficiency. Previous research showed its efficacy against LN229 glioma cells with MMR and MGMT knockdown. Its activity against patient-derived GBM that model post-TMZ recurrent tumors is unclear.
We created MMR-deficient GBM patient-derived xenografts through exposure to TMZ, followed by treatment with additional TMZ or KL-50. We also generated isogenic, MSH6 knockout (KO) patient-derived GBM and tested them for sensitivity to TMZ and KL-50.
KL-50 extended the median survival of mice intracranially engrafted with either patient-derived TMZ-naïve GBM6 or TMZ-naïve GBM12 by 1.75-fold and 2.15-fold, respectively (P < 0.0001). A low dose (4 Gy) of fractionated RT further extended the survival of KL-50-treated GBM12 mice (median survival = 80 days for RT + KL-50 vs. 71 days KL-50 alone, P = 0.018). KL-50 also extended the median survival of mice engrafted with post-TMZ, MMR-deficient GBM6R-m185 (140 days for KL-50 vs. 37 days for vehicle, P < 0.0001). MSH6 KO increased TMZ IC50 for GBM6 and GBM12 cultures by >5-fold and >12-fold for cell death and live cell count outputs, respectively. In contrast, MSH6-KO actually decreased KL-50 IC50 by 10–80%.
KL-50-based compounds are a promising new strategy for the treatment of MGMT-deficient, MMR-deficient GBM that recurs after frontline TMZ.
1) KL-50 treats glioblastoma patient-derived xenografts (PDXs) with or without MMR-deficiency.
2) KL-50 outperforms temozolomide and retains efficacy in treating models of post-TMZ, MMR-deficient PDX.
3) Ionizing radiation enhances the effect of KL-50.
Acquired temozolomide (TMZ) resistance due to DNA mismatch repair (MMR) deficiency is a major problem for glioblastoma (GBM) patients. Until recently, the only options in such cases were nitrosourea agents such as lomustine (CCNU), which have severe side effects. Early studies of KL-50 showed great promise in treating high serum LN229 glioma cells with MMR knockout. In the current study, we extended KL-50 investigations in MMR-deficient and MMR-proficient patient-derived xenograft (PDX) models of isocitrate dehydrogenase (IDH) wild-type GBM, including one recurrent GBM model in which repeated TMZ exposure resulted in adaptive MMR deficiency. The efficacy and tolerability of KL-50 in these settings further support the advancement of this class of therapeutics to clinical trials for patients with post-TMZ recurrent GBM.
Adult-type diffuse gliomas affect nearly 20,000 patients annually in the United States and are nearly always incurable.1,2 The most aggressive form, isocitrate dehydrogenase wild-type glioblastoma (IDHwt GBM) has a particularly grim prognosis, with less than 5% of patients surviving beyond 5 years.3 The DNA alkylating agent temozolomide (TMZ), in combination with maximal surgical resection and radiation therapy (RT), offers some survival benefits, especially in GBMs where the gene encoding the repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT) is suppressed by promoter methylation.4,5 However, GBM commonly recurs even after standard therapy, and few options for such recurrent post-TMZ GBMs exist.6
One of the ways in which GBMs develop TMZ resistance is by acquiring mutations in the genes encoding DNA mismatch repair (MMR) enzymes, mainly MSH2, MSH6, MLH1, and/or PMS2.7 TMZ treatment alkylates DNA bases in tumor cells. When guanine is alkylated at the O6 position, it incorrectly pairs with thymine on the daughter strand of DNA during replication. The cell’s DNA MMR enzymes typically engage in a series of “futile” mismatch repair cycles, then trigger cellular apoptosis when they are unable to repair the incorrect pairing.8,9 Thus, TMZ depends on functional DNA MMR enzymes for effective tumor cell killing. Inactivating mutations in DNA MMR enzyme genes allow TMZ-mediated DNA damage to persist without apoptosis, thus conferring resistance to TMZ. Novel strategies for treating such GBMs are a high clinical priority.
A newly developed imidazotetrazine compound, KL-50, was specifically designed to deposit a 2-fluoroethyl group at the O6 position of guanine in cells lacking MGMT. Subsequent fluoroethylation of guanine leads to the formation of guanine-cytosine adducts and interstrand DNA cross-links (ICLs).10 Since this type of DNA damage cannot be repaired by DNA MMR enzymes, KL-50 leads to cellular apoptosis independent of the MMR pathway. The original study describing KL-50 showed excellent in vitro and in vivo efficacy against LN229 glioma cells in which MGMT and the MMR gene MSH2 had been knocked down.10
We previously published a method to create models of MMR-deficient recurrent GBM by exposing patient-derived xenografts (PDXs) to repeated cycles of TMZ in vivo.11 Here, we employed that technique to evaluate KL-50 against post-TMZ, MMR-impaired GBM PDX, and paired that with in vitro experiments involving isogenic matched parental and MSH6-knockout (KO) GBM cells.
Materials and Methods
Generation of MMR-Deficient PDX
MMR-deficient PDX was generated in accordance with our previously published methods,11 and in accordance with standards set by the Northwestern Institutional Animal Care and Use Committee which approved this project. Briefly, parental IDHwt, EGFRvIII-amplified, partially MGMT-methylated GBM6 PDX and IDHwt, EGFR-amplified, fully MGMT-methylated GBM12 PDX tissues were obtained from the Mayo Clinic PDX repository.12 Tumor spheres were cultured in defined, serum-free media. Nod-scid-gamma (NSG) mice, aged 8–10 weeks, received intracranial injections of 100,000 tumor cells into the right frontal lobes. Equal numbers of male and female mice were engrafted, and mice were randomly assigned to subsequent treatment arms. After 27 days to allow for tumor engraftment, mice received a week-long regimen of TMZ treatment (Sigma-Aldrich #T2577, 2.5 mg/kg daily, intraperitoneal, 5 days weekly). After 1 week of rest, the same TMZ regimen was repeated for 2 additional consecutive week-long cycles. We previously showed that this regimen is sufficient to induce MMR deficiency and TMZ resistance in human GBM PDX.11 After therapy, mice were monitored for tumor recurrence and were euthanized upon becoming symptomatic. Fresh brains were harvested, and tumor tissue was homogenized, mixed with equivalent volumes of Matrigel (Corning #CB40230), and injected into the flanks of a new cohort of NSG mice for propagation and expansion. Flank tissue was then harvested for cryopreservation, snap-freezing, culture, and re-passage of flank tumors.
For CRISPR-generated isogenic in vitro models of mismatch repair-deficient GBM, GBM6 and GBM12 cultures were transduced with lentiviral particles carrying scramble gRNA or 1 of 2 gRNAs targeting the MSH6 gene (gRNA#1: hMSH6 [gRNA#2152] and gRNA#2: hMSH6 [gRNA#2152]), along with a blastidicin resistance gene. These lentiviral particles were purchased from Vectorbuilder. Twenty-four hours post-transduction, cultures were treated with blasticidin. After 10 days of blasticidin treatment, non-transduced controls had completely lost viability while transduced cultures retained viability. MSH6 KO was validated via western blot.
Western Blotting
Western blotting was performed for the DNA MMR proteins (MSH6, MSH2, MLH1, and PMS2), DNA DSB proteins (p-H2A.X and p-p95), along with MGMT, on snap-frozen flank tumor PDX tissue. Briefly, tumor lysate was prepared with RIPA lysis buffer and EDTA-free Protease Inhibitor Cocktail (Thermo Scientific #89901, #87785). Proteins were resolved on 4–20% SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 5% fat-free milk or 5% bovine serum albumin and incubated with primary antibodies (MSH6: mouse, Cell Signaling Technology, #12988; MSH2: mouse, Invitrogen #337900; MLH1: rabbit, Invitrogen #MA5-32041; PMS2: mouse, Invitrogen #MA-26269; MGMT: rabbit, Cell Signaling Technology #2739; p-p95 (S343): rabbit, Cell Signaling Technology #3001; p-H2A.X (S139): rabbit, Cell Signaling Technology #9718; GAPDH: rabbit, Cell Signaling Technology #5174) at concentration of 1:1000 at 4°C overnight, followed by room temperature incubation with respective secondary antibodies (goat anti-rabbit, Cell Signaling Technology #7074; goat anti-mouse, Cell Signaling Technology #7076) at concentration of 1:5000 for 60 min. Membranes were imaged with SuperSignal West Pico PLUS Chemiluminescent reagents (Thermo Scientific #24580) and the BioRad ChemiDoc imaging system.
In Vivo PDX Engraftment and Treatment
Parental PDX (GBM6, GBM12) and MMR-deficient derivative PDX (GBM6R-m185) were used to test KL-50 in vivo. Briefly, tumor spheres were cultured in defined, serum-free media. NSG mice aged 8–10 weeks received intracranial injections of 100,000 tumor cells into right frontal lobes. Groups of mice engrafted with parental PDX received treatment with KL-50, vehicle (10% cyclodextrin plus 5% DMSO in aqueous solution), radiation plus vehicle, or radiation plus KL-50. Groups of mice engrafted with MMR-deficient derivative PDX received treatment with KL-50, TMZ, or vehicle. KL-50 was provided by ModifiBio. Aliquots were dissolved in DMSO, and then diluted with 10% cyclodextrin for treatment. It was administered to mice via oral gavage at a dose of 10 mg/kg of body weight for 5 days a week (M-F) for 3 consecutive weeks, starting on day 11 post-engraftment. TMZ was obtained from Sigma Aldrich (T2577), and was prepared, diluted, and administered similarly, at a dose of 10 mg/kg of body weight. For mice receiving RT, 2.0 Gy of ionizing radiation was administered on days 11 and 18 post-engraftment, respectively.
In Vitro Cytotoxicity and Live Cell Count Assays
GBM6 (MMR+/−), GBM12 (MMR+/−), and GBM6R-m185 (derivative, MMR-deficient GBM6 PDX) glioma spheres were seeded in 12-well tissue culture plates at 50,000 cells per well, using defined, serum-free glioma stem cell media. Cells were treated with TMZ, KL-50, CNNU, or vehicle (DMSO) 24 h after seeding for 5 days. Afterward, cells and media were pelleted via microcentrifugation. The supernatant was aspirated and cells were disassociated with 0.05% trypsin. After trypsin neutralization, cells from each well were centrifuged and re-suspended to a final volume of 50 μL of media. From each tube, 10 μL of suspension was mixed with an equivalent volume of trypan blue, and cell death (percent live cells) and live counts were quantified with a Countess II Automated Cell Counter (Invitrogen #C10283) and chamber slides (Invitrogen #CMQAF1000). For each condition, three biological replicates were assessed in this manner.
Data Processing and Statistical Analysis
Data collection, processing, and statistical analysis were performed with Microsoft Excel and GraphPad Prism V10.1.0. In in vitro dose–response assays, nonlinear regression models were used to determine relative IC50 values. Survival between cohorts was compared via log-rank test. Figures were prepared with GraphPad Prism and Bio Render.
Results
Generation of Post-TMZ, MMR-Deficient PDX
We generated MMR-deficient, post-TMZ PDX in accordance with our previously published methods (Figure 1A).11 Four mice (m184, m185, m197, and m199) engrafted with intracranial GBM6 were treated with 3 cycles of TMZ. The derivative PDX from mouse m185 (designated “GBM6R-m185”) was chosen for further experimentation due to deficiency of all 4 MMR proteins (Figure 1B). MGMT expression was negligible (Figure 1C).
Development and characterization of post-TMZ GBM6 PDX. (A) Illustration of the process for creating post-TMZ, MMR-deficient PDX. Parental GBM6 cells were engrafted intracranially in nod-scid-gamma (NGS) mice, then treated with 3 cycles of TMZ. When tumors recurred and mice succumbed, tumor tissue was implanted into the flank of a new mouse. When flank tumors grew to sufficient size, they were harvested and processed for subsequent in vitro and in vivo therapy testing. (B) MMR protein and (C) MGMT protein expressions in derivative GBM6 PDX flank tissue from 4 mice. Each PDX received 3 cycles of TMZ as an intracranial tumor. MGMT = O-6-methylguanine-DNA methyltransferase; MMR = mismatch repair; PDX = patient-derived xenograft; TMZ = temozolomide
KL-50 in TMZ-Naïve and Post-TMZ MMR-Deficient GBM PDX
KL-50 extended the median survival of mice engrafted with parental TMZ-naïve GBM6 compared to vehicle (57 vs. 32.5 days) and 4 Gy RT (34 days, P < 0.0001 for both comparisons) (Figure 2A). Adding 4 Gy RT to the KL-50 regimen slightly increased median survival further, but this was not statistically significant (63 vs. 57 days, P = 0.32). KL-50 was even more effective in mice engrafted with parental TMZ-naïve GBM12 compared to vehicle (71 vs. 33 days) and RT (36.5 days, P < 0.0001 for both comparisons) (Figure 2B). In the GBM12 PDX model, mice treated with KL-50 + 4 Gy RT survived longer than KL-50 alone (80 days, P = 0.018).
KL-50 in TMZ-naïve and post-TMZ MMR-deficient GBM PDX. (A) Survival for mice engrafted with intracranial parental GBM6 PDX and treated with three cycles of vehicle (M-F for 3 consecutive weeks), KL-50 (10 mg/kg), ionizing radiation (2 separate doses of 2.0 Gy), or radiation plus KL-50. (B) Treatment of parental GBM12 PDX with vehicle, KL-50, RT, or RT plus KL-50. *P < 0.05; ****P < 0.0001. (C) In vitro treatment of GBM6R-m185 with vehicle, TMZ, and KL-50. Cell death (left panel) and live cell counts (right panel) are normalized to vehicle-treated cells. (D) IC50 values generated from in vitro cytotoxicity and live cell count assays with TMZ vs KL-50 in GBM6R-m185. (E) Survival for mice engrafted with intracranial GBM6R-m185 and treated with 3 cycles of vehicle, TMZ (10 mg/kg), or KL-50 (10 mg/kg). GBM = glioblastoma; PDX = patient-derived xenograft; RT = radiation therapy; TMZ = temozolomide. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.
In short-term cultures of post-TMZ, MMR-deficient GBM6R-m185 cells, IC50 analyses showed that KL-50 was 3-fold more effective than TMZ at inducing cytotoxicity and 2-fold more effective at reducing live cell counts (Figure 2C, D). KL-50 extended median survival compared to vehicle (140 vs. 37 days, P < 0.0001) and TMZ (140 vs. 108 days, P = 0.02) (Figure 2E).
KL-50 Overcomes MMR-Mediated TMZ Resistance Similar to CCNU in Isogenic GBM Models of MMR Deficiency
Since TMZ may cause other salient alterations to cells besides MMR inactivation, we generated isogenic models of MMR-proficient and deficient GBM6 and GBM12 cells via CRISPR (Figure 3A). As expected, when MSH6 was inactivated, MSH2 expression also decreased. In both GBM6 and GBM12 cultures, MSH6 KO rendered cells resistant to TMZ-mediated DSBs (as indicated by p-H2A.X protein levels), whereas KL-50 was able to generate DSBs in both MMR-proficient and MMR-deficient contexts (Figure 3B). Phosphorylation of p95, a component of the MRN DSB DNA repair complex, showed similar changes as p-H2A.X.
KL-50 is active against MSH6-deficient GBM cells. (A) MSH6 and MSH2 protein expression in GBM6 and GBM12 PDX transduced with scrambled RNA versus two different guide RNAs for CRISPR knockdown of MSH6. (B) Western blot analysis of DNA DSB markers in GBM6 or GBM12 glioma cultures with and without MSH6 KO. Cultures were treated with 50 µM of TMZ or KL-50 for 5 days. (C) Dose–response curves for TMZ, KL-50, and CCNU treatment of MMR-deficient versus MMR-proficient GBM6 and GBM12 glioma cells. (D) Tabulated IC50 values for dose–response curves shown in panel (B). CCNU = lomustine; GBM = glioblastoma; MMR = mismatch repair; PDX = patient-derived xenograft; TMZ = temozolomide
Using our scramble control and MSH6-KO cultures as models of MMR + and MMR − GBM, we explored the effect of MSH6-KO on the efficacy of TMZ and KL-50 using in vitro cytotoxicity and live cell count assays. Consistent with previous reports of MMR deficiency, MSH6-KO substantially increased resistance to TMZ in both GBM6 and GBM12 models, while KL-50 retained its efficacy in both MMR-proficient and -deficient contexts (Figure 3C, D). In GBM6 cells, MSH6-KO increased TMZ IC50 for cell death and live cell count outputs by 4.9-fold and 12.3-fold, respectively. In GBM12 cells, MSH6-KO increased TMZ IC50 for cell death and live cell count outputs by 11.9-fold and 18.4-fold, respectively. In contrast, the KL-50 IC50 decreased in MSH6-KO GBM6/GBM12 cells compared to parental controls (Figure 3C, D). Unlike KL-50, another ICL-inducing agent, lomustine (CCNU), showed similar activity whether or not MSH6 was knocked out (Figure 3C, D).
Discussion
One of the main challenges in neuro-oncology is treating IDHwt GBM when it recurs after frontline therapy including TMZ. A well-known mechanism of TMZ resistance is the development of inactivating mutations in MMR enzyme-encoding genes, especially in tumors with MGMT promoter methylation that initially show good response to TMZ.7,13–15 Alternative therapies are therefore urgently needed. CCNU has been extensively evaluated as one such alterative, since it causes tumor cell apoptosis independently of MMR enzymes.15 However, CCNU is not a practical option for many patients due to its toxicity profile and narrow therapeutic index.16,17 Immune checkpoint blockade (ICB) has also been tried for treatment of post-TMZ, MMR-deficient gliomas, since such gliomas are hypermutated and neoantigenic. Unfortunately, while ICB works well in gliomas arising in the context of a germline MMR deficiency,18 outcomes in sporadic post-TMZ, MMR deficiency have been equivocal.19,20
To address this therapeutic deficiency, Lin and colleagues developed a family of synthetic DNA-damaging agents for use in post-TMZ GBM.10 Like TMZ, these agents work in MGMT-deficient cells. Unlike TMZ (but like CCNU), they do not require MMR pathways to exert their toxicity. The lead compound in this family, KL-50, previously showed strong activity in high-serum LN229 glioma cells which had been modified by siRNA knockdown of MGMT and the MMR gene MSH2.10
Here, we report successful treatment of MMR-proficient and MMR-deficient IDHwt GBM with KL-50, including a variant with TMZ-induced MMR deficiency, created according to our previously described methods.11 KL-50 extended in vivo survival compared to vehicle and RT monotherapy. Survival extension was more pronounced in mice engrafted with TMZ-naïve GBM12 than with TMZ-naïve GBM6, likely because GBM12 has more MGMT promoter methylation than GBM6.11 Since KL-50 causes ICL and, ultimately, DSBs, we tested its activity in combination with RT. Indeed, a low dose of RT was sufficient to enhance survival when combined with KL-50. In the context of post-TMZ, MMR-deficient GBM, KL-50 was more effective than additional cycles of TMZ, even though the latter still extended mouse survival. This is consistent with our previous observations in post-TMZ, MMR-deficient GBM PDX,11 and may suggest sub-clonal MMR deficiency wherein only some tumor subclones develop complete loss of MMR enzymes. This has been suggested as a common feature of TMZ-driven MMR deficiency in post-therapy gliomas.15,21
Complementary in vitro cytotoxicity and live cell count assays with isogenic MMR-proficient and MMR-deficient GBM PDX models show that not only does KL-50 remain toxic to MMR-deficient GBM cells (unlike TMZ), but that MMR deficiency may enhance such toxicity. The cytotoxic effects of TMZ and KL-50 corresponded well with markers of DNA DSBs, supporting that DSBs generation and repair are central to the mechanism of cytotoxicity. While DNA MMR enzymes repair nicks in DNA caused by alkylating agents, previous research by others showed that MMR enzymes are also involved in the repair of certain kinds of ICLs if they cause sufficient DNA backbone distortion.22 Thus, DNA MMR deficiency may actually increase sensitivity to KL-50, rather than simply having no effect on such sensitivity.
In conclusion, these data demonstrate the potential of KL-50-type compounds in treating patients with post-TMZ, MMR-deficient GBMs, and support the development of a clinical trial aimed at recurrent GBMs.
Acknowledgments
The authors appreciate the support provided by ModifiBio, who donated KL-50 and provided financial support for the experiments reported in this manuscript. The authors also appreciate the support from the Northwestern University veterinary care staff and animal husbandry team.
Funding
This project was supported by funding from ModifiBio. M.M. was supported by National Institutes of Health (NIH) grant F32CA264883. S.A. was supported in part by the Charlie Teo Foundation. T.S. was supported in part by an American Cancer Society postdoctoral fellowship. C.H. was supported by NIH grants R01NS118039 and R01NS117104.
Conflict of interest statement
ModifiBio provided KL-50 and financial support for this project. B.R. and R.B. are affiliated with ModifiBio. Neither B.R./R.B. nor anyone else at ModifiBio had any input regarding the conduct of experiments, interpretation of results, or drafting of the manuscript.
Author contributions
C.H., R.B., and B.R. designed the study. B.R., R.B., and C.H. provided funding and resources. M.M., T.K.S., W.W., R.K.C., and S.A. carried out the experiments. C.H. provided oversight. M.M., T.K.S., and C.H. analyzed data and prepared figures. M.M., T.K.S., and C.H. wrote the manuscript. All authors reviewed and edited the manuscript. M.M. and T.K.S. both contributed equal work as co-first authors.
Data availability
All data referenced in this manuscript is available from the corresponding author upon request.
References
Author notes
Matthew McCord and Thomas Sears contributed equally.
