Failure of senolytic treatment to prevent cognitive decline in a female rodent model of aging.

There are sex differences in vulnerability and resilience to the stressors of aging and subsequent age-related cognitive decline. Cellular senescence occurs as a response to damaging or stress-inducing stimuli. The response includes a state of irreversible growth arrest, the development of a senescence-associated secretory phenotype, and the release of pro-inflammatory cytokines associated with aging and age-related diseases. Senolytics are compounds designed to eliminate senescent cells. Our recent work indicates that senolytic treatment preserves cognitive function in aging male F344 rats. The current study examined the effect of senolytic treatment on cognitive function in aging female rats. Female F344 rats (12 months) were treated with dasatinib (1.2 mg/kg) + quercetin (12 mg/kg) or ABT-263 (12 mg/kg) or vehicle for 7 months. Examination of the estrus cycle indicated that females had undergone estropause during treatment. Senolytic treatment may have increased sex differences in behavioral stress responsivity, particularly for the initial training on the cued version of the watermaze. However, pre-training on the cue task reduced stress responsivity for subsequent spatial training and all groups learned the spatial discrimination. In contrast to preserved memory observed in senolytic-treated males, all older females exhibited impaired episodic memory relative to young (6-month) females. We suggest that the senolytic treatment may not have been able to compensate for the loss of estradiol, which can act on aging mechanisms for anxiety and memory independent of cellular senescence.

Senolytic treatment alleviates doxorubicin-induced chemobrain.

Doxorubicin (Dox), a widely used treatment for cancer, can result in chemotherapy-induced cognitive impairments (chemobrain). Chemobrain is associated with inflammation and oxidative stress similar to aging. As such, Dox treatment has also been used as a model of aging. However, it is unclear if Dox induces brain changes similar to that observed during aging since Dox does not readily enter the brain. Rather, the mechanism for chemobrain likely involves the induction of peripheral cellular senescence and the release of senescence-associated secretory phenotype (SASP) factors and these SASP factors can enter the brain to disrupt cognition. We examined the effect of Dox on peripheral and brain markers of aging and cognition. In addition, we employed the senolytic, ABT-263, which also has limited access to the brain. The results indicate that plasma SASP factors enter the brain, activating microglia, increasing oxidative stress, and altering gene transcription. In turn, the synaptic function required for memory was reduced in response to altered redox signaling. ABT-263 prevented or limited most of the Dox-induced effects. The results emphasize a link between cognitive decline and the release of SASP factors from peripheral senescent cells and indicate some differences as well as similarities between advanced age and Dox treatment.

Targeting KRAS in pancreatic cancer: Emerging therapeutic strategies.

KRAS, a predominant member of the RAS family, is the most frequently mutated oncogene in human pancreatic cancer (∼95% of cases). Mutations in KRAS lead to its constitutive activation and activation of its downstream signaling pathways such as RAF/MEK/ERK and PI3K/AKT/mTOR that promote cell proliferation and provide apoptosis evasion capabilities to cancer cells. KRAS had been considered 'undruggable' until the discovery of the first covalent inhibitor targeting the G12C mutation. While G12C mutations are frequently found in non-small cell lung cancer, these are relatively rare in pancreatic cancer. On the other hand, pancreatic cancer harbors other KRAS mutations such as G12D and G12V. The inhibitors targeting G12D mutation (such as MRTX1133) have been recently developed, whereas those targeting other mutations are still lacking. Unfortunately, KRAS inhibitor monotherapy-associated resistance hinders their therapeutic efficacy. Therefore, various combination strategies have been tested and some yielded promising results, such as combinations with receptor tyrosine kinase, SHP2, or SOS1 inhibitors. In addition, we recently demonstrated that the combination of sotorasib with DT2216 (a BCL-XL-selective degrader) synergistically inhibits G12C-mutated pancreatic cancer cell growth in vitro and in vivo. This is in part because KRAS-targeted therapies induce cell cycle arrest and cellular senescence, which contributes to therapeutic resistance, while their combination with DT2216 can more effectively induce apoptosis. Similar combination strategies may also work for G12D inhibitors in pancreatic cancer. This chapter will review KRAS biochemistry, signaling pathways, different mutations, emerging KRAS-targeted therapies, and combination strategies. Finally, we discuss challenges associated with KRAS targeting and future directions, emphasizing pancreatic cancer.

Effect of peripheral cellular senescence on brain aging and cognitive decline.

We examine similar and differential effects of two senolytic treatments, ABT-263 and dasatinib + quercetin (D + Q), in preserving cognition, markers of peripheral senescence, and markers of brain aging thought to underlie cognitive decline. Male F344 rats were treated from 12 to 18 months of age with D + Q, ABT-263, or vehicle, and were compared to young (6 months). Both senolytic treatments rescued memory, preserved the blood-brain barrier (BBB) integrity, and prevented the age-related decline in hippocampal N-methyl-D-aspartate receptor (NMDAR) function associated with impaired cognition. Senolytic treatments decreased senescence-associated secretory phenotype (SASP) and inflammatory cytokines/chemokines in the plasma (IL-1ß, IP-10, and RANTES), with some markers more responsive to D + Q (TNFα) or ABT-263 (IFNγ, leptin, EGF). ABT-263 was more effective in decreasing senescence genes in the spleen. Both senolytic treatments decreased the expression of immune response and oxidative stress genes and increased the expression of synaptic genes in the dentate gyrus (DG). However, D + Q influenced twice as many genes as ABT-263. Relative to D + Q, the ABT-263 group exhibited increased expression of DG genes linked to cell death and negative regulation of apoptosis and microglial cell activation. Furthermore, D + Q was more effective at decreasing morphological markers of microglial activation. The results indicate that preserved cognition was associated with the removal of peripheral senescent cells, decreasing systemic inflammation that normally drives neuroinflammation, BBB breakdown, and impaired synaptic function. Dissimilarities associated with brain transcription indicate divergence in central mechanisms, possibly due to differential access.

Co-targeting BCL-XL and MCL-1 with DT2216 and AZD8055 synergistically inhibit small-cell lung cancer growth without causing on-target toxicities in mice.

Small-cell lung cancer (SCLC) is an aggressive malignancy with limited therapeutic options. The dismal prognosis in SCLC is in part associated with an upregulation of BCL-2 family anti-apoptotic proteins, including BCL-XL and MCL-1. Unfortunately, the currently available inhibitors of BCL-2 family anti-apoptotic proteins, except BCL-2 inhibitors, are not clinically relevant because of various on-target toxicities. We, therefore, aimed to develop an effective and safe strategy targeting these anti-apoptotic proteins with DT2216 (our platelet-sparing BCL-XL degrader) and AZD8055 (an mTOR inhibitor) to avoid associated on-target toxicities while synergistically optimizing tumor response. Through BH3 mimetic screening, we identified a subset of SCLC cell lines that is co-dependent on BCL-XL and MCL-1. After screening inhibitors of selected tumorigenic pathways, we found that AZD8055 selectively downregulates MCL-1 in SCLC cells and its combination with DT2216 synergistically killed BCL-XL/MCL-1 co-dependent SCLC cells, but not normal cells. Mechanistically, the combination caused BCL-XL degradation and suppression of MCL-1 expression, and thus disrupted MCL-1 interaction with BIM leading to an enhanced apoptotic induction. In vivo, the DT2216 + AZD8055 combination significantly inhibited the growth of cell line-derived and patient-derived xenografts and reduced tumor burden accompanied by increased survival in a genetically engineered mouse model of SCLC without causing appreciable thrombocytopenia or other normal tissue injuries. Thus, these preclinical findings lay a strong foundation for future clinical studies to test DT2216 + mTOR inhibitor combinations in a subset of SCLC patients whose tumors are co-driven by BCL-XL and MCL-1.

Nanovesicle and extracellular polymeric substance synthesis from the remediation of heavy metal ions from soil.

Heavy metal toxicity affects aquatic plants and animals, disturbing biodiversity and ecological balance causing bioaccumulation of heavy metals. Industrialization and urbanization are inevitable in modern-day life, and control and detoxification methods need to be accorded to meet the hazardous environment. Microorganisms and plants have been widely used in the bioremediation of heavy metals. Sporosarcina pasteurii, a gram-positive bacterium that is widely known for its calcite precipitation property in bio-cementing applications has been explored in the study for its metal tolerance ability for the first time. S. pasteurii SRMNP1 (KF214757) can tolerate silver stress to form nanoparticles and can remediate multiple heavy metals to promote the growth of various plants. This astounding property of the isolate warranted extensive examinations to comprehend the physiological changes during an external heavy metal stress condition. The present study aimed to understand various physiological responses occurring in S. pasteuriiSRMNP1 during the metal tolerance phenomenon using electron microscopy. The isolate was subjected to heavy metal stress, and a transmission electron microscope examination was used to analyze the physiological changes in bacteria to evade the metal stress. S. pasteurii SRMNP1 was tolerant against a wide range of heavy metal ions and can withstand a broad pH range (5-9). Transmission Electron Microscopy (TEM) examination of S. pasteurii SRMNP1 followed by 5 mM nickel sulfate treatment revealed the presence of nanovesicles encapsulating nanosized particles in intra and extracellular spaces. This suggests that the bacteria evade the metal stress by converting the metal ions into nanosized particles and encapsulating them within nanovesicles to efflux them through the vesicle budding mechanism. Moreover, the TEM images revealed an excessive secretion of extracellular polymeric substances by the strain to discharge the metal particles outside the bacterial system. S. pasteurii can be foreseen as an effective bioremediation agent with the potential to produce nanosized particles, nanovesicles, and extracellular polymeric substances. This study provides physiological evidence that, besides calcium precipitation applications, S. pasteurii can further be explored for its multidimensional roles in the fields of drug delivery and environmental engineering.

BCL-XL PROTAC degrader DT2216 synergizes with sotorasib in preclinical models of KRASG12C-mutated cancers.

KRAS mutations are the most common oncogenic drivers. Sotorasib (AMG510), a covalent inhibitor of KRASG12C, was recently approved for the treatment of KRASG12C-mutated non-small cell lung cancer (NSCLC). However, the efficacy of sotorasib and other KRASG12C inhibitors is limited by intrinsic resistance in colorectal cancer (CRC) and by the rapid emergence of acquired resistance in all treated tumors. Therefore, there is an urgent need to develop novel combination therapies to overcome sotorasib resistance and to maximize its efficacy. We assessed the effect of sotorasib alone or in combination with DT2216 (a clinical-stage BCL-XL proteolysis targeting chimera [PROTAC]) on KRASG12C-mutated NSCLC, CRC and pancreatic cancer (PC) cell lines using MTS cell viability, colony formation and Annexin-V/PI apoptosis assays. Furthermore, the therapeutic efficacy of sotorasib alone and in combination with DT2216 was evaluated in vivo using different tumor xenograft models. We observed heterogeneous responses to sotorasib alone, whereas its combination with DT2216 strongly inhibited viability of KRASG12C tumor cell lines that partially responded to sotorasib treatment. Mechanistically, sotorasib treatment led to stabilization of BIM and co-treatment with DT2216 inhibited sotorasib-induced BCL-XL/BIM interaction leading to enhanced apoptosis in KRASG12C tumor cell lines. Furthermore, DT2216 co-treatment significantly improved the antitumor efficacy of sotorasib in vivo. Collectively, our findings suggest that due to cytostatic activity, the efficacy of sotorasib is limited, and therefore, its combination with a pro-apoptotic agent, i.e., DT2216, shows synergistic responses and can potentially overcome resistance.

Overcoming Gemcitabine Resistance in Pancreatic Cancer Using the BCL-XL-Specific Degrader DT2216.

Pancreatic cancer is the third most common cause of cancer-related deaths in the United States. Although gemcitabine is the standard of care for most patients with pancreatic cancer, its efficacy is limited by the development of resistance. This resistance may be attributable to the evasion of apoptosis caused by the overexpression of BCL-2 family antiapoptotic proteins. In this study, we investigated the role of BCL-XL in gemcitabine resistance to identify a combination therapy to more effectively treat pancreatic cancer. We used CRISPR-Cas9 screening to identify the key genes involved in gemcitabine resistance in pancreatic cancer. Pancreatic cancer cell dependencies on different BCL-2 family proteins and the efficacy of the combination of gemcitabine and DT2216 (a BCL-XL proteolysis targeting chimera or PROTAC) were determined by MTS, Annexin-V/PI, colony formation, and 3D tumor spheroid assays. The therapeutic efficacy of the combination was investigated in several patient-derived xenograft (PDX) mouse models of pancreatic cancer. We identified BCL-XL as a key mediator of gemcitabine resistance. The combination of gemcitabine and DT2216 synergistically induced cell death in multiple pancreatic cancer cell lines in vitro In vivo, the combination significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice compared with the individual agents in pancreatic cancer PDX models. Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-XL and concomitant suppression of MCL-1 by gemcitabine. Our results suggest that DT2216-mediated BCL-XL degradation augments the antitumor activity of gemcitabine and their combination could be more effective for pancreatic cancer treatment.

Senescence-associated hyper-activation to inflammatory stimuli in vitro.

Aging is associated with an increased susceptibility to adverse inflammatory conditions such as sepsis and cytokine storm. We hypothesized that senescent cells (SnCs) play a central role in this age-associated pathology in part due to their expression of the senescence-associated secretory phenotype (SASP), which may prime SnCs to inflammatory stimulation. To test this hypothesis, we examined the expression of various inflammatory cytokines and chemokines at the levels of gene transcription and protein production in various SnCs in vitro in response to lipopolysaccharide (LPS), interleukin-1ß (IL1ß), and tumor necrosis factor α (TNFα) stimulation. We found that SnCs not only expressed higher basal levels of various inflammatory cytokines and chemokines as a manifestation of the SASP, but more importantly exhibited hyper-activation of the induction of a variety of inflammatory mediators in response to LPS, IL1ß and TNFα stimulation as compared with non-SnCs. This senescence-associated hyper-activation is likely mediated in part via the p38MAPK (p38) and NFκB pathways because LPS stimulation elicited significantly higher levels of p38 phosphorylation and NFκB p65 nuclear translation in SnCs when compared to their non-senescent counterparts and inhibition of these pathways with losmapimod (a p38 specific inhibitor) and BMS-345541 (a selective NFκB inhibitor) attenuated LPS-induced expression of IL6, TNFα, CCL5, and IL1ß mRNA in SnCs. These findings suggest that SnCs may play an important role in the age-related increases in the susceptibility to developing an exacerbated inflammatory response and highlight the potential to use senotherapeutics to ameliorate the severity of various devastating inflammatory conditions in the elderly.

Cellular senescence in lymphoid organs and immunosenescence.

Immunosenescence is a multi-faceted phenomenon at the root of age-associated immune dysfunction. It can lead to an array of pathological conditions, including but not limited to a decreased capability to surveil and clear senescent cells (SnCs) and cancerous cells, an increased autoimmune responses leading to tissue damage, a reduced ability to tackle pathogens, and a decreased competence to illicit a robust response to vaccination. Cellular senescence is a phenomenon by which oncogene-activated, stressed or damaged cells undergo a stable cell cycle arrest. Failure to efficiently clear SnCs results in their accumulation in an organism as it ages. SnCs actively secrete a myriad of molecules, collectively called senescence-associated secretory phenotype (SASP), which are factors that cause dysfunction in the neighboring tissue. Though both cellular senescence and immunosenescence have been studied extensively and implicated in various pathologies, their relationship has not been greatly explored. In the wake of an ongoing pandemic (COVID-19) that disproportionately affects the elderly, immunosenescence as a function of age has become a topic of great importance. The goal of this review is to explore the role of cellular senescence in age-associated lymphoid organ dysfunction and immunosenescence, and provide a framework to explore therapies to rejuvenate the aged immune system.

DT2216-a Bcl-xL-specific degrader is highly active against Bcl-xL-dependent T cell lymphomas.

BACKGROUND: Patients with advanced T cell lymphomas (TCLs) have limited therapeutic options and poor outcomes in part because their TCLs evade apoptosis through upregulation of anti-apoptotic Bcl-2 proteins. Subsets of TCL cell lines, patient-derived xenografts (PDXs), and primary patient samples depend on Bcl-xL for survival. However, small molecule Bcl-xL inhibitors such as ABT263 have failed during clinical development due to on-target and dose-limiting thrombocytopenia. METHODS: We have developed DT2216, a proteolysis targeting chimera (PROTAC) targeting Bcl-xL for degradation via Von Hippel-Lindau (VHL) E3 ligase, and shown that it has better anti-tumor activity but is less toxic to platelets compared to ABT263. Here, we examined the therapeutic potential of DT2216 for TCLs via testing its anti-TCL activity in vitro using MTS assay, immunoblotting, and flow cytometry and anti-TCL activity in vivo using TCL cell xenograft and PDX model in mice. RESULTS: The results showed that DT2216 selectively killed various Bcl-xL-dependent TCL cells including MyLa cells in vitro. In vivo, DT2216 alone was highly effective against MyLa TCL xenografts in mice without causing significant thrombocytopenia or other toxicity. Furthermore, DT2216 combined with ABT199 (a selective Bcl-2 inhibitor) synergistically reduced disease burden and improved survival in a TCL PDX mouse model dependent on both Bcl-2 and Bcl-xL. CONCLUSIONS: These findings support the clinical testing of DT2216 in patients with Bcl-xL-dependent TCLs, both as a single agent and in rational combinations.

Discovery of IAP-recruiting BCL-XL PROTACs as potent degraders across multiple cancer cell lines.

Targeting BCL-XL via PROTACs is a promising strategy in reducing BCL-XL inhibition associated platelet toxicity. Recently, we reported potent BCL-XL PROTAC degraders that recruit VHL or CRBN E3 ligase. However, low protein expression or mutation of the responsible E3 ligase has been known to result in decreased protein degradation efficiency of the corresponding PROTACs. To overcome these mechanisms of resistance, PROTACs based on recruiting alternative E3 ligases could be generated. Thus, we designed and synthesized a series of PROTACs that recruit IAP E3 ligases for BCL-XL degradation. Among those PROTACs, compound 8a efficiently degrades BCL-XL in malignant T-cell lymphoma cell line MyLa 1929 while CRBN-based PROTACs that have high potency in other cancer cell lines show compromised potency, likely due to the low CRBN expression. Moreover, compared with the parent compound ABT-263, PROTAC 8a shows comparable cell killing effects in MyLa 1929 cells whereas the on-target platelet toxicity is significantly reduced. Our findings expand the anti-tumor spectra of BCL-XL degraders and further highlight the importance of selecting suitable E3 members to achieve effective cellular activity.

Using proteolysis-targeting chimera technology to reduce navitoclax platelet toxicity and improve its senolytic activity.

Small molecules that selectively kill senescent cells (SCs), termed senolytics, have the potential to prevent and treat various age-related diseases and extend healthspan. The use of Bcl-xl inhibitors as senolytics is largely limited by their on-target and dose-limiting platelet toxicity. Here, we report the use of proteolysis-targeting chimera (PROTAC) technology to reduce the platelet toxicity of navitoclax (also known as ABT263), a Bcl-2 and Bcl-xl dual inhibitor, by converting it into PZ15227 (PZ), a Bcl-xl PROTAC, which targets Bcl-xl to the cereblon (CRBN) E3 ligase for degradation. Compared to ABT263, PZ is less toxic to platelets, but equally or slightly more potent against SCs because CRBN is poorly expressed in platelets. PZ effectively clears SCs and rejuvenates tissue stem and progenitor cells in naturally aged mice without causing severe thrombocytopenia. With further improvement, Bcl-xl PROTACs have the potential to become safer and more potent senolytic agents than Bcl-xl inhibitors.

Inhibition of USP7 activity selectively eliminates senescent cells in part via restoration of p53 activity.

The accumulation of senescent cells (SnCs) is a causal factor of various age-related diseases as well as some of the side effects of chemotherapy. Pharmacological elimination of SnCs (senolysis) has the potential to be developed into novel therapeutic strategies to treat these diseases and pathological conditions. Here we show that ubiquitin-specific peptidase 7 (USP7) is a novel target for senolysis because inhibition of USP7 with an inhibitor or genetic depletion of USP7 by RNA interference induces apoptosis selectively in SnCs. The senolytic activity of USP7 inhibitors is likely attributable in part to the promotion of the human homolog of mouse double minute 2 (MDM2) ubiquitination and degradation by the ubiquitin-proteasome system. This degradation increases the levels of p53, which in turn induces the pro-apoptotic proteins PUMA, NOXA, and FAS and inhibits the interaction of BCL-XL and BAK to selectively induce apoptosis in SnCs. Further, we show that treatment with a USP7 inhibitor can effectively eliminate SnCs and suppress the senescence-associated secretory phenotype (SASP) induced by doxorubicin in mice. These findings suggest that small molecule USP7 inhibitors are novel senolytics that can be exploited to reduce chemotherapy-induced toxicities and treat age-related diseases.