Rosenfeld, Steven S.

Abstract We have shown that members of the non-muscle myosin II (NMII) family of molecular motors are non-redundant drivers of both invasion and proliferation in glioblastoma (GBM), consistent with the canonical roles NMII isoforms play in cell motility and cytokinesis. However, we have also found that NMII also has a non-canonical role by regulating oncogenic signaling pathways relevant to GBM pathogenesis. This unexpected finding has led us to identify and characterize MT-125, a highly specific small molecule inhibitor of the two predominant NMII isoforms in GBM (NMIIA and NMIIB), and we have studied its effects in pre-clinical GBM models. MT-125 has high brain penetrance and retention; an excellent safety profile in rodents and canines with no changes in metabolic or hematologic indices after prolonged administration; is well-tolerated with daily dosing for at least 85 consecutive days; blocks GBM invasion and cytokinesis, consistent with the canonical roles of NMII; and prolongs median survival by 35% as a single agent in murine GBM models. MT-125 increases signaling along both the PDGFR- and MAPK-driven pathways through a mechanism that involves the upregulation of reactive oxygen species, leading to oncogene addiction to these pathways. As a consequence, MT-125 is synthetically lethal when combined with FDA-approved, CNS-permeant inhibitors of PDGFR and mTOR in vitro. Combining MT-125 with sunitinib, a PDGFR inhibitor, or paxalisib, a combined PI3 Kinase/mTOR inhibitor, in vivo doubles median survival in a highly aggressive murine orthotopic GBM model. Furthermore, combining MT-125 with sunitinib produces tumor free remissions of >100 days--2.5-fold greater than median survival of either drug alone—in approximately 40% of mice. Our results provide a powerful rationale for developing NMII targeting strategies to treat cancer and demonstrate that MT-125 has strong clinical potential for the treatment of GBM.

ImportanceImplementing multidisciplinary teams for treatment of complex brain tumors needing awake craniotomies is associated with significant costs. To date, there is a paucity of analysis on the cost utility of introducing advanced multidisciplinary standardized teams to enable awake craniotomies.ObjectiveTo assess the cost utility of introducing a standardized program of awake craniotomies.Design, Setting, and ParticipantsA retrospective economic evaluation was conducted at Mayo Clinic Florida. All patients with single, unilateral lesions who underwent elective awake craniotomies between January 2016 and December 2021 were considered eligible for inclusion. The economic perspective of the health care institution and a time horizon of 1 year were considered. Data were analyzed from October 2022 to May 2023.ExposureTreatment with an awake craniotomy before standardization (2016-2018) compared with treatment with awake craniotomy after standardization (2018-2021).Main Outcomes and MeasuresPatient demographics, perioperative, and postoperative outcomes, including length of stay, intensive care (ICU) admission, extent of resection, readmission rates, and 1-year mortality were compared between patients undergoing surgery before and after standardization. Direct medical costs were estimated from Medicare reimbursement rates for all billed procedures. A cost-utility analysis was performed considering differences in direct medical costs and in 1-year mortality within the periods before and after standardization of procedures. Uncertainty was explored in probability sensitivity analysis.ResultsA total of 164 patients (mean [SD] age, 49.9 [15.7] years; 98 [60%] male patients) were included in the study. Of those, 56 underwent surgery before and 108 after implementation of procedure standardization. Procedure standardization was associated with reductions in length of stay from a mean (SD) of 3.34 (1.79) to 2.46 (1.61) days (difference, 0.88 days; 95% CI, 0.33-1.42 days; P = .002), length of stay in ICU from a mean (SD) of 1.32 (0.69) to 0.99 (0.90) nights (difference, 0.33 nights; 95% CI, 0.06-0.60 nights; P = .02), 30-day readmission rate from 14% (8 patients) in the prestandardization cohort to 5% (5 patients) (difference, 9%; 95% CI, 19.6%-0.3%; P = .03), while extent of resection and intraoperative complication rates were similar between both cohorts. The standardized protocol was associated with mean (SD) savings of $7088.80 ($12 389.50) and decreases in 1-year mortality (dominant intervention). This protocol was found to be cost saving in 75.5% of all simulations in probability sensitivity analysis.Conclusions and RelevanceIn this economic evaluation of standardization of awake craniotomy, there was a generalized reduction in length of stay, ICU admission time, and direct medical costs with implementation of an optimized protocol. This was achieved without compromising patient outcomes and with similar extent of resection, complication rates, and reduced readmission rates.

Abstract Sequencing efforts have identified key oncogenic drivers and transcriptomic subtypes of high-grade gliomas (HGGs), but this knowledge has not resulted in individualized therapy or improved patient outcomes. Here, we used a functional genomics approach combining integrated genomic/transcriptomic analyses with patient-derived 3D micro-cancers (mCancers) to rapidly identify individualized therapeutic vulnerabilities and determine predictive response biomarkers. 30 primary/recurrent HGGs, including 20 glioblastomas, 5 astrocytomas, and 5 oligodendrogliomas, were included for drug testing. mCancers recapitulated the structural and molecular alterations, cellularity, and heterogeneity, of parental patient tumors. A total of 119 single-agent drugs and 183 drug combinations were tested, while a pre-selected drug panel comprising 25 glioma-relevant treatment strategies was tested across all cases. In general, mCancer efficacy corresponded well to historical responses to previously tested agents (EGFR, HDAC inhibitors, etc.). Combined, strong efficacy (i.e. >70% growth inhibition at Cmax) of single treatments targeting deregulated receptor tyrosine kinase (RTK), p53/apoptosis, cell cycle, DNA damage repair, IDH, or telomere maintainance pathways, was observed in 14 out of 30 cases. Surprisingly, transcriptional signatures, but not specific genetic alterations, were found to stratify drug responses. For example, mCancers enriched for the most aggressive mesenchymal (MES) and glycolytic/plurimetabolic (GPM) transcriptional subtypes exhibited stronger response to the BET inhibitor JQ1 (and other clinical BET inhibitors) and the DNA-PKcs/mTOR dual inhibitor CC-115. This therapeutic vulnerability was associated with enrichment of YAP/TAZ signaling, and the glioblastoma stem cell G-STEM signature. Notably, individual combination strategies were effective across tumors, with strong responses ranging from 38% to 77% of all tumors tested. Overall, this study underscores the value of functional genomics in identifying novel vulnerabilities of HGGs and associated response biomarkers. It also highlights the need for further clinical validation of several novel therapies and especially drug combination strategies with strong efficacy in the ex vivo setting.

Abstract Tumor cells require the mitotic spindle to drive chromosome separation in metaphase, which has made the spindle a compelling target for development of anti-mitotics. However, most of these spindle inhibitors target microtubules and can thereby be highly neurotoxic. This has motivated development of targeted anti-mitotics that inhibit non-microtubule components of the spindle, including kinesins and mitotic kinases. These drugs are not neurotoxic and prolong survival in tumor patients, but resistance limits their efficacy. We reported (Kenchappa et al., Cell Reports, 2022) that resistance to one of these—ispinesib, a highly specific inhibitor of the mitotic kinesin Kif11—requires two functions of STAT3: activation of transcription in the nucleus and of oxidative phosphorylation in the mitochondria. Inhibiting both functions reverses ispinesib resistance. Furthermore, combining ispinesib with a CNS-permeant STAT3 inhibitor significantly prolongs survival over ispinesib alone in both immunocompetent and PDX GBM models. We now report that resistance to two other targeted anti-mitotics—alisertib (Aurora Kinase inhibitor) and volasertib (Polo Like Kinase inhibitor)—works similarly and can likewise be reversed with STAT3 inhibitors. For all three of these anti-mitotics, resistance goes hand-in-hand with development of therapy-induced senescence (TIS) in approximately half of GBM cells. TIS cells secrete a variety of soluble factors, referred to as the senescence associated secretory phenotype (SASP), which stimulate stemness, angiogenesis, and immunosuppression. We propose that: 1) TIS cells require STAT3 for survival, and that they in turn maintain a drug resistant state by suppressing mitosis in the remaining, non senescent cells through components of the SASP; and 2) by killing TIS cells, STAT3 inhibitors restore anti-mitotic sensitivity in the remaining, non-senescent tumor cells. We propose that this STAT3/TIS model of resistance may also apply to how resistance develops to other GBM therapies, and may guide similar translational approaches to enhancing therapeutic efficacy.

KIF20A is a critical kinesin for cell division and a promising anti-cancer drug target. The mechanisms underlying its cellular roles remain elusive. Interestingly, unusual coupling between the nucleotide- and microtubule-binding sites of this kinesin-6 has been reported, but little is known about how its divergent sequence leads to atypical motility properties. We present here the first high-resolution structure of its motor domain that delineates the highly unusual structural features of this motor, including a long L6 insertion that integrates into the core of the motor domain and that drastically affects allostery and ATPase activity. Together with the high-resolution cryo-electron microscopy microtubule-bound KIF20A structure that reveals the microtubule-binding interface, we dissect the peculiarities of the KIF20A sequence that influence its mechanochemistry, leading to low motility compared to other kinesins. Structural and functional insights from the KIF20A pre-power stroke conformation highlight the role of extended insertions in shaping the motor's mechanochemical cycle. Essential for force production and processivity is the length of the neck linker in kinesins. We highlight here the role of the sequence preceding the neck linker in controlling its backward docking and show that a neck linker four times longer than that in kinesin-1 is required for the activity of this motor.

Kinesin-5 crosslinks and slides apart microtubules to assemble, elongate, and maintain the mitotic spindle. Kinesin-5 is a tetramer, where two N-terminal motor domains are positioned at each end of the motor, and the coiled-coil stalk domains are organized into a tetrameric bundle through the bipolar assembly (BASS) domain. To dissect the function of the individual structural elements of the motor, we constructed a minimal kinesin-5 tetramer (mini-tetramer). We determined the x-ray structure of the extended, 34-nm BASS domain. Guided by these structural studies, we generated active bipolar kinesin-5 mini-tetramer motors from Drosophila melanogastor and human orthologues which are half the length of native kinesin-5. We then used these kinesin-5 mini-tetramers to examine the role of two unique structural adaptations of kinesin-5: 1) the length and flexibility of the tetramer, and 2) the C-terminal tails which interact with the motor domains to coordinate their ATPase activity. The C-terminal domain causes frequent pausing and clustering of kinesin-5. By comparing microtubule crosslinking and sliding by mini-tetramer and full-length kinesin-5, we find that both the length and flexibility of kinesin-5 and the C-terminal tails govern its ability to crosslink microtubules. Once crosslinked, stiffer mini-tetramers slide antiparallel microtubules more efficiently than full-length motors.

Abstract Diffuse gliomas are aggressive brain tumors that lack effective therapies. Despite advances in the molecular characterization of gliomas, targeted treatments have failed to improve patient outcomes to date. Here, we employed a functional genomics approach to rapidly identify therapeutic vulnerabilities and provide a strategy to individualize patient treatment. Our process combined comprehensive genomic and transcriptomic tumor profiling, custom algorithms and visualization software for data integration, and preclinical 3-dimensional ex vivo models (mcancers) for drug screening to assess response to therapeutic agents targeting specific molecular alterations. A total of 32 cases, including 5 oligodendrogliomas (1 primary and 4 recurrent), 5 astrocytomas (1 primary and 4 recurrent), and 22 glioblastomas (5 primary and 17 recurrent), were included in the study. Immunohistochemistry and integrated genomics confirmed that mcancers maintain key molecular alterations, including mutations, chromosomal rearrangements and focal amplifications, cellularity, and heterogeneity, faithfully recapitulating the original tumor. A total of 119 single drugs and 183 combinations were tested, including a panel of 26-brain penetrant targeted agents (18 single drugs and 8 combinations) that was curated to target common glioma oncogenic pathways and was tested across all cases. A strong correlation was observed between therapeutic vulnerabilities of glioma mcancers to targeted agents (EGFR, MET, PDGFR, HDAC inhibitors, etc.) and clinical experience. Single agent activity of panel compounds in the ex vivo setting was generally poor, with only 35% mCancers exhibiting > 70% growth inhibition at Cmax. In contrast, combination strategies, especially ones including epigenetic drugs, were significantly more effective (affecting 22 out of 32 mCancers), underscoring the molecular complexity of gliomas and the need for clinical trials testing combination treatments. Our data identified a number of novel combination treatments with significant efficacy across glioma subtypes and molecular profiles, suggesting that our functional genomics pipeline can guide treatment selection and inform clinical trial enrollment for gliomas.

Abstract While the allure of targeted therapies in oncology has been their high degree of specificity and potency for key tumor drivers, they have been disappointing in glioblastoma (GBM), even for drugs that are blood brain barrier permeable and CNS retained. This point is highlighted by the experience with mitotic spindle inhibitors, drugs which block the G2M transition and induce mitotic catastrophe—a phenotype characterized by cell enlargement and polyploidy that leads to apoptotic cell death. We have shown that one of these, a potent inhibitor of the mitotic kinesin Kif11 (ispinesib), is highly active against GBM tumor initiating cells and prolongs survival in murine models of this disease. However, tumors eventually progress, reflecting the development of drug resistance. Although ispinesib resistant GBM cells develop mitotic catastrophe, they become highly resistant to the apoptosis that typically follows and continue to proliferate. We find that this apoptosis resistance requires phosphorylation of the transcription factor STAT3 at two residues—Y705 and S727. Phosphorylation of Y705, mediated by SRC kinase, translocates STAT3 to the nucleus where it induces transcription of anti-apoptotic proteins. Phosphorylation at S727, mediated by EGFR, translocates STAT3 to the mitochondria where it blocks release of cytochrome c—the penultimate effector in apoptosis. Simultaneously inhibiting both SRC and EGFR with FDA-approved, CNS permeant inhibitors reverses this resistance and significantly prolongs survival in ispinesib-treated GBM-bearing mice. Furthermore, we find that resistance to several other mitotic inhibitors also utilizes this STAT3-driven mechanism and can likewise be reversed with combined EGFR and SRC inhibition. Thus, our work demonstrates how a promising therapeutic approach, which has been disappointing in GBM, can in fact be rendered effective by anticipating and prospectively treating ab initio the mechanism that drives treatment resistance.