Cyclin-dependent kinases 4 and 6 (CDK4/6) play critical roles in the G1 to S checkpoint of the cell cycle and have been shown to be overactive in several human cancers. Small-molecule inhibitors of ...CDK4/6 have demonstrated significant efficacy against many solid tumors. Since CDK4/6 inhibition is thought to induce cell cycle arrest at the G1/S checkpoint, much interest has been focused on combining CDK4/6 inhibitors with cytotoxic agents active against the S or M phase of the cell cycle to enhance therapeutic efficacy. However, it remains unclear how best to combine these two classes of drugs to avoid their potentially antagonistic effects. Here, we test various combinations of highly selective and potent CDK4/6 inhibitors with commonly used cytotoxic drugs in several cancer cell lines derived from lung, breast and brain cancers, for their cell-killing effects as compared to monotherapy. All combinations, either concurrent or sequential, failed to enhance cell-killing effects. Importantly, in certain schedules, especially pre-treatment with a CDK4/6 inhibitor, combining these drugs resulted in reduced cytotoxicity of cytotoxic agents. These findings urge cautions when combining these two classes of agents in clinical settings.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Learning a novel motor skill is associated with well characterized structural and functional plasticity in the rodent motor cortex. Furthermore, neuroimaging studies of visuomotor learning in humans ...have suggested that structural plasticity can occur in white matter (WM), but the biological basis for such changes is unclear. We assessed the influence of motor skill learning on WM structure within sensorimotor cortex using both diffusion MRI fractional anisotropy (FA) and quantitative immunohistochemistry. Seventy-two adult (male) rats were randomly assigned to one of three conditions (skilled reaching, unskilled reaching, and caged control). After 11 d of training, postmortem diffusion MRI revealed significantly higher FA in the skilled reaching group compared with the control groups, specifically in the WM subjacent to the sensorimotor cortex contralateral to the trained limb. In addition, within the skilled reaching group, FA across widespread regions of WM in the contralateral hemisphere correlated significantly with learning rate. Immunohistological analysis conducted on a subset of 24 animals (eight per group) revealed significantly increased myelin staining in the WM underlying motor cortex in the hemisphere contralateral (but not ipsilateral) to the trained limb for the skilled learning group versus the control groups. Within the trained hemisphere (but not the untrained hemisphere), myelin staining density correlated significantly with learning rate. Our results suggest that learning a novel motor skill induces structural change in task-relevant WM pathways and that these changes may in part reflect learning-related increases in myelination.
The organization of forelimb representation areas of the monkey, cat, and rat motor cortices has been studied in depth, but its characterization in the mouse lags far behind. We used intracortical ...microstimulation (ICMS) and cytoarchitectonics to characterize the general organization of the C57BL/6 mouse motor cortex, and the forelimb representation in more detail. We found that the forelimb region spans a large area of frontal cortex, bordered primarily by vibrissa, neck, shoulder, and hindlimb representations. It included a large caudal forelimb area, dominated by digit representation, and a small rostral forelimb area, containing elbow and wrist representations. When the entire motor cortex was mapped, the forelimb was found to be the largest movement representation, followed by head and hindlimb representations. The ICMS-defined motor cortex spanned cytoarchitecturally identified lateral agranular cortex (AGl) and also extended into medial agranular cortex. Forelimb and hindlimb representations extended into granular cortex in a region that also had cytoarchitectural characteristics of AGl, consistent with the primary motor-somatosensory overlap zone (OL) characterized in rats. Thus, the mouse motor cortex has homologies with the rat in having 2 forelimb representations and an OL but is distinct in the predominance of digit representations.
BackgroundDiffuse midline glioma (DMG) is a universal fatal glial brain cancer in children. We tested our novel multilamellar mRNA lipid particle aggregate vaccine (RNA-LPA, IND19304—Sayour),1 a ...tumor-agnostic treatment platform that encapsulates tumor specific RNA and delivers the payload in a highly immunogenic fashion, as an approach to treating this currently incurable cancer.MethodsUsing the K2 DMG model,2 we implant H3K27M-expressing DMG cells into the 4th ventricle of P1-P3 neonatal C57BL/6 mice. RNA-LPA generated from predicated human H3K27M epitopes or total-tumor mRNA are administered intravenously beginning at day 35. We performed multiparameter 3D geospatial fluorescent microscopy to characterize mRNA transduction. Immunologic responses to treatment were evaluated by multiparameter flow cytometry, microscopy, and cytokine profiling.ResultsMice developed clinical neurological signs of disease by day 30–35. RNA-LPAs targeting human H3K27M epitopes were found to be immunogenic in wild-type mice. Intriguingly, nonspecific enhanced green fluorescent protein (eGFP)-RNA-LPAs resulted in statistically significant survival benefits compared to mice treated with empty LPs. However, tumor-specific RNA-LPAs (either H3K27M-specific or total tumor mRNA-derived) also enhanced survival and additionally resulted in a subset of mice with long-term survival. This survival benefit was observed despite the development of clinical hydrocephalus in mice treated with RNA-LPAs. 3D microscopy established that tumors demonstrated invasive disease and microvascular erosion in mice. We found that mRNA transduces fibroblastic reticular cells (FRCs) in the spleen and lymph nodes, prompting widespread immune activation. Treatment with RNA-LPA led to massive increases in production inflammatory cytokines (i.e. TNF-α) and chemokines (i.e. CCL2), which led to recruitment of the majority of circulating monocytes and lymphocytes to secondary lymphoid organs.ConclusionsRNA-LPAs extend survival in our highly aggressive DMG model, including curative outcomes in cohorts treated with either total tumor or H3K27M RNA-LPs. These data suggest that RNA-LPs are capable of stimulating host adaptive immune responses against established DIPG tumors. Signs of hydrocephalus in treated mice may indicate pseudoprogression due to immunologic response, yet mice were frequently able to survive this development. Future studies will further characterize the immunologic response in these mice and support expansion of our existing IND for a multi-institutional phase I clinical trial for children with DMG, who currently have no curative options.AcknowledgementsWe appreciate funding from the ChadTough Defeat DIPG Foundation and the DIPG/DMG Research Funding Alliance. John Ligon and Elias Sayour contributed equally and are co-senior authors.ReferencesMendez-Gomez H, DeVries A, Castillo P, Stover B, Qdaisat S, Von Roemling C, Ogando-Rivas E, Weidert F, McGuiness J, Zhang D, Chung MC, Li D, Zhao C, Marconi C, Campaneria Y, Chardon-Robles J, Grippin A, Karachi A, Thomas N, Huang J, Milner R, Sahay B, Sawyer WG, Ligon JA, Silver N, Simon E, Cleaver B, Wynne K, Hodik M, Molinaro A, Guan J, Kellish P, Doty A, Lee J-H, Carrera-Justiz S, Rahman M, Gatica S, Mueller S, Prados M, Ghiaseddin A, Mitchell DA, Sayour EJ. mRNA aggregates harness danger response for potent cancer immunotherapy. medRxiv. 2023:2023.03.12.23287108. doi: 10.1101/2023.03.12.23287108.Misuraca KL, Cordero FJ, Becher OJ. Pre-Clinical Models of Diffuse Intrinsic Pontine Glioma. Front Oncol. 2015;5:172. doi: 10.3389/fonc.2015.00172. PubMed PMID: 26258075; PMCID: PMC4513210.Ethics ApprovalWork approved under UF IACUC 202200000375
► Plasticity of the young motor map is transient despite long-term skill practice. ► Aged mice can learn a skilled reaching task in a similar way to young mice. ► The organization and plasticity of ...motor representations is altered with aging. ► Reaching behavior and motor map plasticity are at least partially dissociable. ► Motor map reorganization seems to be a transient stage in motor learning.
Movement representations in the motor cortex can reorganize to support motor skill learning during young adulthood. However, little is known about how motor representations change during aging or whether their change is influenced by continued practice of a skill after it is learned. We used intracortical microstimulation to characterize the organization of the forelimb motor cortex in young and aged C57/BL6 mice after short (2–4weeks) or long (8weeks) durations of training on a skilled reaching task or control procedures. In young mice, a short duration of reach training increased the area of proximal forelimb movement representations at the expense of distal representations. Following a longer training duration, ratios of proximal to distal movements returned to baseline, even with ongoing practice and skill maintenance. However, lingering changes were evident in thresholds for eliciting distal forelimb movements, which declined over the longer training period. In aged mice, movement representations and movement thresholds failed to change after either duration of training. Furthermore, there was an age-related loss of digit representations and performance decrements on other sensorimotor tests. Nevertheless, in quantitative measures of reaching success, aged mice learned and performed the skilled reaching task at least as well as younger mice. These results indicate that experience-driven topographical reorganization of motor cortex varies with age, as well as time, and is partially dissociable from behavioral performance. They also support an enduring capacity to learn new manual skills during aging, even as more youthful forms of cortical plasticity and sensorimotor function are lost.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Background. Recent evidence suggests that motor training may be beneficial for slowing the onset of motor impairments in Parkinson’s disease (PD). Objective. To examine the impact of targeted ...rehabilitation on limb motor and cranial motor function and the corresponding corticospinal and corticobulbar circuits in a rodent model of PD. Methods. Baseline performance of limb (reaching) and cranial (licking) motor function were established prior to and 6 weeks following unilateral intrastriatal 6-hydroxydopamine (6-OHDA) infusions. Animals then received 6 weeks of limb motor rehabilitation (LMR) or cranial motor rehabilitation (CMR), after which motor performance was reassessed. Intracortical microstimulation (ICMS) was used to generate motor maps of corresponding corticospinal (forelimb) and corticobulbar (tongue) movement representations within the motor cortex ipsilateral to the 6-OHDA infusion. Quantitative tyrosine hydroxylase (TH) immunohistochemistry was performed to determine levels of striatal TH depletion in 6-OHDA animals using near infrared densitometry. Results. (1) unilateral intrastriatal dopamine depletion impaired both reaching accuracy and lick force; (2) targeted LMR ameliorated impairments in reaching performance; however, CMR did not improve lick force impairments; (3) unilateral dopamine depletion significantly reduced forelimb but not tongue motor map topography; (4) LMR partially restored forelimb motor maps, whereas CMR did not alter tongue motor maps; and (5) significant correlations were observed between skilled reaching accuracy, forelimb motor map area, and TH depletion, but no relationships were revealed for cranial motor function, motor maps, or TH depletion. Conclusions: These data demonstrate dissociation between striatal dopamine depletion, limb versus cranial motor function, and targeted motor rehabilitation in a rodent model of PD.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
► Unilateral dopamine depletion impairs limb and cranial motor function. ► Limb motor impairments are more severe than cranial motor impairments. ► Only limb motor function was correlated to degree ...of dopamine depletion. ► Suggesting differential sensitivities to dopamine depletion in a rodent model of PD.
The present study determined the differential effects of unilateral striatal dopamine depletion on cranial motor versus limb motor function. Forty male Long Evans rats were first trained on a comprehensive motor testing battery that dissociated cranial versus limb motor function and included: cylinder forepaw placement, single pellet reaching, vermicelli pasta handling; sunflower seed opening, pasta biting acoustics, and a licking task. Following baseline testing, animals were randomized to either a 6-hydroxydopamine (6-OHDA) (n=20) or control (n=20) group. Animals in the 6-OHDA group received unilateral intrastriatal 6-OHDA infusions to induce striatal dopamine depletion. Six-weeks following infusion, all animals were re-tested on the same battery of motor tests. Near infrared densitometry was performed on sections taken through the striatum that were immunohistochemically stained for tyrosine hydroxylase (TH). Animals in the 6-OHDA condition showed a mean reduction in TH staining of 88.27%. Although 6-OHDA animals were significantly impaired on all motor tasks, limb motor deficits were more severe than cranial motor impairments. Further, performance on limb motor tasks was correlated with degree of TH depletion while performance on cranial motor impairments showed no significant correlation. These results suggest that limb motor function may be more sensitive to striatal dopaminergic depletion than cranial motor function and is consistent with the clinical observation that therapies targeting the nigrostriatal dopaminergic system in Parkinson's disease are more effective for limb motor symptoms than cranial motor impairments.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Rehabilitation-dependent motor recovery after cerebral ischemia is associated with functional reorganization of residual cortical tissue. Recovery is thought to occur when remaining circuitry ...surrounding the lesion is “retrained” to assume some of the lost function. This reorganization is in turn supported by synaptic plasticity within cortical circuitry and manipulations that promote plasticity may enhance recovery. Activation of the cAMP/CREB pathway is a key step for experience-dependent neural plasticity. Here we examined the effects of the prototypical phosphodiesterase inhibitor 4 (PDE4) rolipram and a novel PDE inhibitor (HT-0712), known to enhance cAMP/CREB signaling and cognitive function, on restoration of motor skill and cortical function after focal cerebral ischemia. Adult male rats were trained on a skilled reaching task to establish a baseline level of motor performance. Intracortical microstimulation was then used to derive high-resolution maps of forelimb movement representations within the caudal forelimb area of motor cortex contralateral to the trained paw. A focal ischemic infarct was created within approximately 30% of the caudal forelimb area. The effects of administering either rolipram or the novel PDE4 inhibitor HT-0712 during rehabilitation on motor recovery and restoration of movement representations within residual motor cortex were examined. Both compounds significantly enhanced motor recovery and induced an expansion of distal movement representations that extended beyond residual motor cortex. The expansion beyond the initial residual cortex was not observed in vehicle injected controls. Furthermore, the motor recovery seen in the HT-0712 animals was dose dependent. Our results suggest that PDE4 inhibitors during motor rehabilitation facilitate behavioral recovery and cortical reorganization after ischemic insult to levels significantly greater than that observed with rehabilitation alone.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Abstract
Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults despite aggressive chemoradiotherapy. Tumor Treating Fields (TTFields) was recently approved in ...combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM patients. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules’ assembly, leading to disrupted chromosomal segregation, integrity and stability. In many patients, a transient stage of increased peritumoral edema is often observed early in the course of TTFields treatment followed subsequently by objective radiographic responses, suggesting that a major component of therapeutic efficacy by TTFields may be an immune mediated process. However, the mechanism underlying these observations remains unclear. Here we report results on a panel of GBM cell lines treated with TTFields at the clinically approved frequency of 200 kHz using an in vitro TTFields system. Our data showed 24 hrs TTFields-treated GBM cells had a significantly higher rate (19.9% vs. 4.3%, p=0.0032) of micronuclei structures released into the cytoplasm as a result of TTFields-induced chromosomal instability. Nearly 40% of these micronuclei were co-localized with two upstream dsDNA sensors: absent in melanoma 2 (AIM2)andInterferon (IFN)-inducible proteinCyclic GMP-AMP synthase (cGAS), compared to absence of co-localization in untreated cells. TTFields-activated micronuclei-dsDNA sensor complexes led to i) induction of pyroptotic cell death, as measured by a specific LDH release assay, and through AIM2-recruited caspase1 and cleavage of pyroptosis-specific Gasdermin D; and ii) activation of STING pathway components including Type I IFNs and pro-inflammatory cytokines downstream of the NFκB pathway. GBM cell-specific shRNA depletion of either AIM2 or STING or both in a co-culture experiment of bone marrow cells or splenocytes with supernanants obtained from knockdown GBM cells was able to reverse the inducement of immune cells. These results provide compelling evidence that TTFields function as an activator of the immune system in GBM cells, and a strong rationale for combining TTFields with immunotherapy aimed at augmenting an anti-tumor immune response such as immune checkpoint inhibitors.
Note: This abstract was not presented at the meeting.
Citation Format: Dongjiang Chen, Nagheme Thomas, David D. Tran. TTFields induces immunogenic cell death and STING pathway activation through cytoplasmic double-stranded DNA in glioblastoma cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3280.
While limb motor deficits of Parkinson's disease are well characterized, the effects of striatal dopamine depletion on the motor cortex is poorly understood. We therefore aimed to 1) examine the ...effects of striatal dopamine depletion on forelimb function and cortical motor map topography and 2) explore potential relationships between forelimb function and cortical movement representations in an animal model of PD. Twenty-four male Long Evans rats were randomized to control or 6-hydroxydopamine (6-OHDA) groups. Animals in the 6-OHDA group underwent four unilateral 6-OHDA infusions into the striatum to induce striatal dopamine depletion. Four weeks later, animals were tested on a comprehensive battery of behavioral limb motor tasks followed by intracortical microstimulation to derive high-resolution topographic maps of forelimb movement representations. Standard tyrosine hydroxylase (TH) immunohistochemistry was performed and near infrared densitometry analysis utilized to assess TH depletion. Unilateral striatal dopamine depletion induced significant reductions in limb motor function that were reflected neurophysiologically as a reduction in cortical forelimb movement representations. Voluntary forelimb use, pasta handling, sunflower seed manipulation, and forelimb motor maps were all significantly impaired in 6-OHDA animals. A positive correlation was observed between forelimb function and motor map size, as well as two negative correlations between TH depletion with 1) motor map size and 2) forelimb function. The results clearly show how dysfunction within the basal ganglia thalamocortical loop resulting from nigrostriatal dopamine depletion disrupts corticospinal function.
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