Showing papers by "Philip L. Lorenzi published in 2023"
TL;DR: In this paper , an inhaled therapeutic that stimulates lung epithelial defenses to protect mice against pneumonia in a manner that depends on production of reactive oxygen species (ROS) was identified.
Abstract: Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infections an urgent need. We have previously shown that manipulating the lungs’ intrinsic host defenses by therapeutic delivery of a unique dyad of pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODNs) with mitochondrial voltage-dependent anion channel 1 (VDAC1) without dependence on Toll-like receptor 9 (TLR9). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), enhances mitochondrial membrane potential (ΔΨm), and differentially modulates ETC complex activities. These combined effects promote leak of electrons from ETC complex III, resulting in superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy that has the potential to broadly protect patients against pneumonia during periods of peak vulnerability without reliance on currently available antibiotics. Author Summary Pneumonia is a major cause of death worldwide. Increasing antibiotic resistance and expanding immunocompromised populations continue to enhance the clinical urgency to find new strategies to prevent and treat pneumonia. We have identified a novel inhaled therapeutic that stimulates lung epithelial defenses to protect mice against pneumonia in a manner that depends on production of reactive oxygen species (ROS). Here, we report that the induction of protective ROS from lung epithelial mitochondria occurs following the interaction of one component of the treatment, an oligodeoxynucleotide, with the mitochondrial voltage-dependent anion channel 1. This interaction alters energy transfer between the mitochondria and the cytosol, resulting in metabolic reprogramming that drives more electrons into the electron transport chain, then causes electrons to leak from the electron transport chain to form protective ROS. While antioxidant therapies are endorsed in many other disease states, we present here an example of therapeutic induction of ROS that is associated with broad protection against pneumonia without reliance on administration of antibiotics.
TL;DR: Baek et al. as discussed by the authors investigated the effects of conventional TNBC chemotherapies singly and in standard combinations, on mitochondrial translation and electron transport chain (ETC) formation in human TNBC cells and PDX models using metabolomic and proteomic profiling.
Abstract:
BACKGROUND: Nearly 50% of patients with triple negative breast cancer (TNBC) treated with neoadjuvant chemotherapy (NACT) retain residual tumors resulting in high rates of metastatic relapse and poor overall survival. Residual tumors surviving NACT (Adriamycin plus cyclophosphamide; AC) were found to undergo a metabolic transition to heightened mitochondrial oxidative phosphorylation (oxphos; PMID: 30996079). Pharmacologic inhibition of mitochondrial electron transport chain (ETC) complex I with IACS-010759 (PMID: 29892070) had enhanced efficacy in residual, rather than treatment-naïve, tumors of orthotopic patient-derived xenograft (PDX) models. Our analyses of mitochondrial structure and function in human TNBC cell lines revealed differing adaptations in residual cells surviving treatment with conventional NACT agents. While DNA-damaging chemotherapies (e.g.Adriamycin, carboplatin) induced mitochondrial fusion and oxphos, taxanes (e.g.paclitaxel, docetaxel) induced mitochondrial fragmentation and reduced oxphos (Baek et al., Biorxiv Doi 10.1101/2022.02.25.481996). The mechanistic basis of these mitochondrial adaptations is not yet understood. The mitochondrial ETC consists of 92 proteins, 13 of which are encoded in the mitochondrial genome (mtDNA) and translated by the mitoribosome, while the remaining are encoded by the nuclear genome (nDNA), translated by the cytoribosome, and inserted into the inner mitochondrial membrane by accessory proteins, namely Oxidase (Cytochrome C) Assembly 1-Like (OXA1L). Disruption of OXA1L in mammalian cells has been shown to affect the levels and activity of ETC complexes I, III, IV, and V, and thus diminish oxphos. We aim to determine whether mitochondrial translation and OXA1L activity represent therapeutic vulnerabilities to overcome pro-survival metabolic adaptations in chemoresistant TNBC thereby augmenting treatment response. METHODS: Weare evaluating the effects of conventional TNBC chemotherapies singly, and in standard combinations, on mitochondrial translation and ETC formation in human TNBC cells and PDX models(PIM001-P, WHIM14, BCM15116) using metabolomic and proteomic profiling. To perturb these processes genetically, we knocked down (KD) OXA1Lwith siRNA. We are complementing these studies pharmacologically using conventional antibiotics, such as tigecycline, as previous studies showed they inhibit mitochondrial translation in breast and other cancers (PMID: 25625193). These studies will reveal whether OXA1L and mitochondrial translation are required for metabolic adaption and chemotherapy resistance of residual TNBC cells. PDX preclinical trials based on our published residual tumor testing schema (PMID: 30996079), will reveal whether the sequential combination of NACT followed by tigecycline can effectively perturb residual tumor relapse. RESULTS: Proteomic profiling of longitudinally harvested PDX tumors demonstrates substantial disruption of mitochondria-and nuclear-encoded ETC components in residual vs. treatment-naïve tumors. Interestingly, these patterns are distinct between different chemotherapy treatments, with an increase of ETC components in carboplatin-treated residual tumors compared to a decrease in docetaxel-treated residual tumors. Western blot analyses of human cell lines show OXA1LKD perturbs levels of both nuclear-and mitochondria-encoded ETC components. Preliminary findings suggest OXA1LKD increases sensitivity to chemotherapies in human TNBC cell lines. Finally, tigecycline effectively inhibits TNBC cell growth. We next will evaluate whether residual cells not killed by conventional chemotherapies have enhanced tigecycline susceptibility. CONCLUSION: These data suggest targeting mitochondrial translation may be a promising approach to overcome pro-survival metabolic adaptations in residual TNBC cells not killed by conventional chemotherapies.
Citation Format: Mariah J. Berner, Lily Baek, Junegoo Lee, Philip L. Lorenzi, Mei Leng, Alexander B. Saltzman, Anna Malovannaya, Lacey E. Dobrolecki, Christina Sallas, Michael T. Lewis, Gloria V. Echeverria. Investigating the role of mitochondrial protein translation in the metabolic adaptation of chemoresistant triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-10.
TL;DR: In this article , the authors found that depleting FAD pools in mice with a vitamin B2-deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease.
Abstract: Flavin adenine dinucleotide (FAD) interacts with flavoproteins to mediate oxidation-reduction reactions required for cellular energy demands. Not surprisingly, mutations that alter FAD binding to flavoproteins cause rare inborn errors of metabolism (IEMs) that disrupt liver function and render fasting intolerance, hepatic steatosis, and lipodystrophy. In our study, depleting FAD pools in mice with a vitamin B2-deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease. Integrated discovery approaches revealed B2D tempered fasting activation of target genes for the nuclear receptor PPARα, including those required for gluconeogenesis. We also found PPARα knockdown in the liver recapitulated B2D effects on glucose excursion and fatty liver disease in mice. Finally, treatment with the PPARα agonist fenofibrate activated the integrated stress response and refilled amino acid substrates to rescue fasting glucose availability and overcome B2D phenotypes. These findings identify metabolic responses to FAD availability and nominate strategies for the management of organic acidemias and other rare IEMs.
TL;DR: In this article , a study in pancreatic ductal adenocarcinoma (PDAC) found that cellular and mitochondrial lipid composition influence cancer cell sensitivity to pharmacological inhibition of electron transport chain complex I.
Abstract: Mitochondria are hubs where bioenergetics, redox homeostasis, and anabolic metabolism pathways integrate through a tightly coordinated flux of metabolites. The contributions of mitochondrial metabolism to tumor growth and therapy resistance are evident, but drugs targeting mitochondrial metabolism have repeatedly failed in the clinic. Our study in pancreatic ductal adenocarcinoma (PDAC) finds that cellular and mitochondrial lipid composition influence cancer cell sensitivity to pharmacological inhibition of electron transport chain complex I. Profiling of patient-derived PDAC models revealed that monounsaturated fatty acids (MUFAs) and MUFA-linked ether phospholipids play a critical role in maintaining ROS homeostasis. We show that ether phospholipids support mitochondrial supercomplex assembly and ROS production; accordingly, blocking de novo ether phospholipid biosynthesis sensitized PDAC cells to complex I inhibition by inducing mitochondrial ROS and lipid peroxidation. These data identify ether phospholipids as a regulator of mitochondrial redox control that contributes to the sensitivity of PDAC cells to complex I inhibition.
TL;DR: Chen et al. as mentioned in this paper proposed that targeting PAPSS1 in PTEN/PAPSS2-null prostate cancer can generate cancer-specific vulnerabilities while leaving normal cells undisturbed.
Abstract:
Sulfation of proteins, carbohydrates, lipids, and xenobiotics is an essential post-translational modification (PTM) process thought to play critical roles in diverse biological processes ranging from detoxification, cell signaling, and extracellular matrix architecture, to immune modulation. Sulfation is accomplished by the universal sulfate donor, PAPS (3'-Phosphoadenosine-5'-phosphosulfate), which is synthesized by bifunctional enzymes PAPSS1 and PAPSS2 (PAPS synthases). The PAPSS2 gene situates near PTEN and is frequently deleted with PTEN across cancer types. Approximately 20% of prostate cancer patients exhibit loss of PTEN, and ~50% of these cases also sustain a loss of PAPSS2. However, the loss of PAPSS2 appears to be tolerated and possibly compensated by its functionally redundant paralogue, PAPSS1, located on chromosome 4q24. The functional redundancy between PAPSS1 and PAPSS2 suggests that these two genes may be collateral lethality pair provided that sulfation is essential for cancer cell viability. Thus, we hypothesize that targeting PAPSS1 in PTEN/PAPSS2-null prostate cancer can generate cancer-specific vulnerabilities while leaving normal cells undisturbed. To assess this possibility, knockdown and knockout of PAPSS1 in cell lines of PAPSS2-null and PAPSS2-wildtype background were generated to characterize cell viability in vitro and tumor formation in vivo. PAPS and APS, an intermediary product of the sulfation pathway, are measured to verify that no alternative pathways for sulfate donors exist and that the co-extinction of PAPSS1/2 eliminates all avenues of generating sulfate donors. Combined extinction of PAPSS1/2 across multiple cancer cell lines was shown to be tolerated in vitro, and recurrent changes in morphology were observed. Loss of sulfation verified by the disappearance of sulfotyrosine and mass spectrometry measurements of PAPS and APS are pending. Our In vitro results surprisingly indicate that a major PTM, like sulfation is entirely dispensable for cancer cell viability under normal culture conditions. However, PAPSS1/2-null cell lines demonstrated a profound delay in tumor formation and prolonged survival, suggesting that sulfation may be required for stromal and innate immune modulation.
Citation Format: Ko-Chien Chen, Yonhong Liu, Chenchu Lin, Er-Yen (Nick) Yen, Francesca Citron, Xingdi Ma, Tan Lin, Philip Lorenzi, Florian Muller, Ronald DePinho. Sulfation is required for prostate cancer xenograft tumor formation but is dispensable for cell viabilityin vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6053.
TL;DR: Baek et al. as mentioned in this paper showed that TNBC cells metabolically adapt to the stress of NACT by upregulating lipid metabolic pathways, providing highly energetic molecules that can be utilized to drive oxphos in chemoresistant TNBC.
Abstract:
Background: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype for which limited targeted therapies are available. Therefore, conventional chemotherapy remains the backbone of standard neoadjuvant treatment (NACT) for TNBC patients. Unfortunately, ~45% of patients will have substantial residual tumor burden post neoadjuvant chemotherapy, leading to poor prognoses (PMID: 28135148). Recently, it has been demonstrated that mitochondrial oxidative phosphorylation (oxphos) is upregulated and is a therapeutic vulnerability in chemoresistant TNBC (PMID: 30996079; Baek et al., BioRxiv doi.org/10.1101/2022.02.25.481996). However, mechanisms driving increased oxphos in chemoresistant TNBC are not understood. Upregulated fatty acid (FA) metabolism is a common adaptation in tumors, providing an energy source through fatty acid β-oxidation (FAO), and promoting lipid accumulation after fatty acid synthesis (FAS) when energy needs are met. Chemotherapy can induce oxidative stress through the generation of reactive oxygen species. Cancer cells adapt to these damaging molecules by increasing de novo lipogenesis, resulting in the accumulation of lipid droplets (LDs) in the cytosol (PMID: 32782526, 20876798). We hypothesize that TNBC cells metabolically adapt to the stress of NACT by upregulating lipid metabolic pathways, providing highly energetic molecules that can be utilized to drive oxphos in chemoresistant TNBC. Methods: Using orthotopic patient-derived xenograft (PDX) models of TNBC (PIM001-P, PMID: 30996079, HCI-010, PMID: 22019887; WHIM14, PMID:24055055), we are measuring protein levels of fatty acid synthase (FASN) in vehicle tumors vs residual tumors surviving treatment with the standard front-line neoadjuvant chemotherapy regimens (Adriamycin plus cyclophosphamide (AC), docetaxel, carboplatin, or docetaxel+carboplatin) using immunohistochemistry (IHC). Vectra 3 microscopy (Akoya) is being used to quantify tumor cell-specific staining. We complemented our IHC analysis with reverse-phase protein array (RPPA). To assess LD accumulation in residual PDX tumors, we conducted transmission electron microscopy (TEM). To complement these PDX studies, we modeled the residual tumor metabolic state in cultured human TNBC cells. Following treatment with the IC50 of standard chemotherapeutic agents (AC, carboplatin, paclitaxel, docetaxel), we assessed oxphos by measuring oxygen consumption rate (OCR) using a Seahorse Bioanalyzer (Agilent). Further, we tested LD accumulation using LipidTOX staining. In ongoing studies, we are measuring incorporation of 13C palmitate into the tricarboxylic acid cycle (TCA) prior to and following chemotherapy treatments to assess if lipids fuel mitochondrial metabolism in residual TNBC cells. Results/Discussion: IHC in the PIM001-P PDX model after in vivo AC treatment revealed increased levels of FASN in post-AC residual tumors compared to the treatment-naive tumors. Further, key proteins involved in fatty acid synthesis, FASN and Acetyl-CoA carboxylase, were significantly increased in residual PIM001-P cells that survived AC compared to vehicle by RPPA. TEM analysis of the HCI-010 PDX revealed significantly more LDs in carboplatin-treated tumors compared to vehicle. This finding was supported by increased LDs observed in TNBC cell lines treated with NACT compared to vehicle in our LipidTOX analyses. Taken together, these data indicate that NACT induces increased expression of key lipid metabolism proteins and accumulation of cytosolic LDs. Our future experiments will reveal if chemoresistant TNBC cells preferentially utilize and incorporate lipids into the tricarboxylic acid cycle, in turn driving oxphos. These data have the potential to provide rationale for the incorporation of FAO/LD inhibitors in sequential combinations with conventional chemotherapies to more effectively kill TNBC cells that are chemo-refractory.
Citation Format: Katherine E. Pendleton, Mokryun L. Baek, Junegoo Lee, Lin Tan, Hannah L. Johnson, Lacey E. Dobrolecki, James P. Barrish, Michael T. Lewis, Philip L. Lorenzi, Fabio Stossi, Gloria V. Echeverria. Lipid accumulation in residual triple negative breast cancer cells surviving chemotherapy treatment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-15.
TL;DR: In this article , the authors found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and loss of Bacteroides ovatus from the microbiome.
Abstract: Abstract Acute gastrointestinal intestinal GVHD (aGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation, and the intestinal microbiota is known to impact on its severity. However, an association between treatment response of aGI-GVHD and the intestinal microbiota has not been well-studied. In a cohort of patients with aGI-GVHD (n=37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and loss of Bacteroides ovatus from the microbiome. In a mouse model of carbapenem-aggravated GVHD, introducing Bacteroides ovatus reduced severity of GVHD and improved survival. Bacteroides ovatus reduced degradation of colonic mucus by another intestinal commensal, Bacteroides thetaiotaomicron, via its ability to metabolize dietary polysaccharides into monosaccharides, which then inhibit mucus degradation by Bacteroides thetaiotaomicron and reduce GVHD-related mortality.
TL;DR: In this article , the authors describe the antitumor activity and underlying molecular mechanisms of a novel Na+/K+-ATPase inhibitor RX108 in human hepatocellular carcinoma (HCC) cells and its xenograft model.
Abstract: The poor prognosis and limited therapeutic options for human hepatocellular carcinoma (HCC), the most common form of liver cancer, highlight the urgent need to identify novel therapeutic modalities. Here we describe the antitumor activity and underlying molecular mechanisms of a novel Na+/K+-ATPase inhibitor RX108 in human HCC cells and its xenograft model. RX108 dose-dependently inhibited HCC cell proliferation in vitro and tumor growth in a xenograft mouse model, and that the inhibition was associated with induction of apoptosis. Mechanistically, RX108 significantly downregulated alanine serine cysteine transporter 2 (ASCT2) protein expression and reduced glutamine and glutamate concentration in HCC cells and tumors. Additionally, RX108 exposure led to a significant decrease in cell energy metabolism in Huh7 and Hep3B cells, including decreased levels of glutathione, NADH, NADPH, and mitochondrial respiration oxygen consumption rate (OCR). Furthermore, HCC cells exhibited evidence of glutamine addiction; the antiproliferative effect of RX108 was dependent on glutamine transport. Clinically, elevated ASCT2 mRNA expression in HCCs was associated with unfavorable survival. Taken together, these findings reveal a novel approach to target glutamine metabolism through inhibiting Na+/K+-ATPase and provide a rationale for using RX108 to treat HCC in patients whose tumors express ASCT2 at high levels. RX108 is currently under clinical development.
TL;DR: In this paper , a CRISPR/Cas9 screening strategy was used to identify the mitochondrial DNA repair protein DNA2, whose loss of function suppresses MM cells' ability to overcome ILF2−induced DNA damage.
Abstract: DNA damage resistance is a major barrier to effective DNA-damaging therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense oligonucleotide (ASO) therapy targeting ILF2, a DNA damage regulator that is overexpressed in 70% of MM patients whose disease has progressed after standard therapies have failed. Here, we show that MM cells undergo an adaptive metabolic rewiring and rely on oxidative phosphorylation to restore energy balance and promote survival in response to DNA damage activation. Using a CRISPR/Cas9 screening strategy, we identified the mitochondrial DNA repair protein DNA2, whose loss of function suppresses MM cells’ ability to overcome ILF2 ASO−induced DNA damage, as being essential to counteracting oxidative DNA damage and maintaining mitochondrial respiration. Our study revealed a novel vulnerability of MM cells that have an increased demand for mitochondrial metabolism upon DNA damage activation. STATEMENT OF SIGNIFICANCE Metabolic reprogramming is a mechanism through which cancer cells maintain survival and become resistant to DNA-damaging therapy. Here, we show that targeting DNA2 is synthetically lethal in myeloma cells that undergo metabolic adaptation and rely on oxidative phosphorylation to maintain survival after DNA damage activation.
TL;DR: In this article , the authors showed that the breast tumor suppressor gene SIM2 promotes mitochondrial oxidative phosphorylation (OXPHOS) using breast cancer cell line models and found that SIM2s functions not as a transcription factor but localizes to mitochondria and directly interacts with the MRC to facilitate functional supercomplex (SC) formation.
Abstract: Abstract Dysregulation of cellular metabolism is a hallmark of breast cancer progression and is associated with metastasis and therapeutic resistance. Here, we show that the breast tumor suppressor gene SIM2 promotes mitochondrial oxidative phosphorylation (OXPHOS) using breast cancer cell line models. Mechanistically, we found that SIM2s functions not as a transcription factor but localizes to mitochondria and directly interacts with the mitochondrial respiratory chain (MRC) to facilitate functional supercomplex (SC) formation. Loss of SIM2s expression disrupts SC formation through destabilization of MRC Complex III, leading to inhibition of electron transport, although Complex I (CI) activity is retained. A metabolomic analysis showed that knockout of SIM2s leads to a compensatory increase in ATP production through glycolysis and accelerated glutamine-driven TCA cycle production of NADH, creating a favorable environment for high cell proliferation. Our findings indicate that SIM2s is a novel stabilizing factor required for SC assembly, providing insight into the impact of the MRC on metabolic adaptation and breast cancer progression.
TL;DR: In this article , a combination of trametinib and vincristine was used to improve the efficacy of MEK inhibitors in patients with metastatic colorectal cancer (mCRC).
Abstract: Mutations in KRAS are found in more than 50% of tumors from patients with metastatic colorectal cancer (mCRC). However, direct targeting of most KRAS mutations is difficult; even the recently developed KRASG12C inhibitors failed to show significant benefit in patients with mCRC. Single agents targeting MEK, a downstream mediator of RAS, have also been ineffective in CRC. To identify drugs that can enhance the efficacy of MEK inhibitors we performed unbiased high-throughput screening using CRC spheroids. We used trametinib as the anchor drug and examined combinations of trametinib with the National Cancer Institute Approved Oncology Library version 5. The initial screen, and following focused validation screens, identified vincristine as being strongly synergistic with trametinib. In vitro, the combination strongly inhibited cell growth, reduced clonogenic survival, and enhanced apoptosis compared to monotherapies in multiple KRAS-mutant CRC cell lines. Furthermore, this combination significantly inhibited tumor growth, reduced cell proliferation, and increased apoptosis in multiple KRAS-mutant patient-derived xenograft mouse models. In vivo studies using drug doses that reflect clinically achievable doses demonstrated that the combination was well tolerated by mice. We further determined that the mechanism underlying the synergistic effect of the combination was due to enhanced intracellular accumulation of vincristine associated with MEK inhibition. The combination also significantly decreased p-mTOR levels in vitro, indicating that it inhibits both RAS-RAF-MEK and PI3K-AKT-mTOR survival pathways. Our data thus provide strong evidence that the combination of trametinib and vincristine represents a novel therapeutic option to be studied in clinical trials for patients with KRAS-mutant mCRC.