DDRE-24. ARF4-MEDIATED RETROGRADE TRAFFICKING PROMOTES CHEMORESISTANCE IN GBM

Abstract Glioblastoma (GBM) is the most common type of adult malignant brain tumor, with a median survival of only 21 months. This is partly due to the high rate of resistance to conventional therapy, including temozolomide (TMZ), leading to recurrence rates close to 100%. To identify the unknown genes driving the development of this resistance, we performed a genome-wide CRISPR knockout screen comparing a DMSO-treated population with a TMZ-treated population over 14 days. Results showed significant enrichment of ~200 novel genes and pathways. From this list, we identified 4 previously unstudied genes showing significant elevations in RNA expression (p< 0.05) when comparing recurrent and primary tumors in patient datasets, along with significant survival benefits corresponding to low gene expression (p< 0.05). Validation experiments in vitro showed significant elevations in RNA and protein expression in multiple patient-derived xenografts (PDX) lines during TMZ-treated conditions, while knocking out these genes also resulted in significantly heightened sensitivity to TMZ (p< 0.01). We investigated one particularly enriched gene, ARF4, known to be involved in retrograde trafficking. With previous studies showing that ARF4 is upregulated under ER stress, we first confirmed the increased expression of ER stress markers during TMZ treatment to explain the increased expression of ARF4 during treatment. Further investigation via live-cell imaging also showed a consequent increase in retrograde trafficking in TMZ-treated cells, as evidenced by significantly increased trafficking of transferrin receptors, a retrograde transport marker, as well as EGFR, known to play a role in promoting chemoresistance through strengthened DNA repair response. ARF4-overexpressed GBM cells similarly showed increased trafficking of transferrin receptors and EGFR to the nucleus, while ARF4-knockdowns showed decreased trafficking and nuclear EGFR expression. Ultimately, our CRISPR-Cas9 screen has identified a promising therapeutic target, ARF4, which may drive GBM’s robust resistance to chemotherapy through increased retrograde trafficking of chemoresistance-promoting nuclear EGFR.

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ARF4-mediated Retrograde Trafficking Drives Chemoresistance in Glioblastoma

10.1101/2021.07.18.451328 ◽

2021 ◽

Author(s):

Shreya Budhiraja ◽

Shivani Baisiwala ◽

Ella Perrault ◽

Sia Cho ◽

Khizar Nandoliya ◽

...

Keyword(s):

Retrograde Transport ◽

Common Type ◽

High Rate ◽

Recurrence Rates ◽

Retrograde Trafficking ◽

Genome Wide ◽

Promising Therapeutic Target ◽

Significant Survival ◽

A Genome ◽

Resistance To Chemotherapy

Glioblastoma (GBM) is the most common type of adult malignant brain tumor, with a median survival of only 21 months. This is partly due to the high rate of resistance to conventional therapy, including temozolomide (TMZ), leading to recurrence rates close to 100%. It still remains unknown what drives the development of this resistance. To identify the unknown genes driving the development of this resistance, we performed a genome-wide CRISPR knockout screen comparing a DMSO-treated population with a TMZ-treated population over 14 days. We identified 4 previously unstudied genes – ARF4, PLAA, SPTLC1, and PIGK – that showed significant elevations in expression in recurrent tumors in patient datasets, along with significant survival benefits corresponding to low gene expression. Further investigation of ARF4, known to be involved in retrograde trafficking, allowed us to identify a mechanism of resistance that is mediated by increased retrograde transport of EGFR into the nucleus. Ultimately, our CRISPR-Cas9 screen has identified a promising therapeutic target, ARF4, which may drive GBM's high resistance to chemotherapy.

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Reconstitution of Retrograde Transport from the Golgi to the ER In Vitro

The Journal of Cell Biology ◽

10.1083/jcb.143.3.589 ◽

1998 ◽

Vol 143 (3) ◽

pp. 589-599 ◽

Cited By ~ 92

Author(s):

Anne Spang ◽

Randy Schekman

Keyword(s):

Vesicle Trafficking ◽

Retrograde Transport ◽

Snare Proteins ◽

Reporter Protein ◽

In Vitro System ◽

Retrograde Trafficking ◽

Simple Set ◽

Membrane Bound

Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone α-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369–377). Retrieval depends on the HDEL sequence; the α-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo.

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CBIO-12. KAT5 ACTIVITY CONTROLS GLIOBLASTOMA CELL CYCLE DYNAMICS AND TUMOR GROWTH

Neuro-Oncology ◽

10.1093/neuonc/noab196.112 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi29-vi29

Author(s):

Anca Mihalas ◽

Heather Feldman ◽

Sonali Arora ◽

John Bassett ◽

Anoop Patel ◽

...

Keyword(s):

Cell Cycle ◽

Tumor Cells ◽

Genetically Engineered ◽

Lower Grade ◽

Current Standard ◽

Primary Tumors ◽

A Genome ◽

G0 Phase ◽

Cell Cycle Dynamics

Abstract Current standard of care therapy for glioblastoma (GB) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, non-cycling, quiescent-like states (G0 phase cells) are present in both normal tissue and tumors and play important roles in maintaining heterogeneity and cellular hierarchies. The presence of quiescent-like/G0 states therefore represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. However, it remains largely unclear to what degree or by what mechanisms tumor cells enter into or exit from quiescent-like states. To gain insight into how GB cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GB stem-like cells (GSCs) harboring a G0 reporter construct, which is stabilized when cells enter a G0-like state. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, including KAT5 itself. Remarkably, we show that knockout of KAT5 in vitro and in vivo dramatically increases G0 subpopulations in GSC cultures and GSC-induced tumors. Using genetically engineered GSC harboring KAT5 under the control of a Doxycyclin-titratable promoter, we establish that incrementally down regulating KAT5 activity is sufficient to slow cell cycle dynamics causing a build-up G0-like cells; and that partial inhibition of KAT5 leads to extended (mouse) patient survival. Further, in primary tumors, cell-based KAT5 activity assays revealed that high grade tumors harbor larger cell subpopulations with higher KAT5 activity than lower grade tumors. In summary, our results suggest that Tip60/KAT5 activity plays key roles in G0 ingress/egress for GBM tumors, may contribute to tumor progression, and may provide novel therapeutic opportunities.

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Adenylate Kinase 2 Is a Selective Dependency in NSD2-High Multiple Myeloma

Blood ◽

10.1182/blood-2020-141266 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-31

Author(s):

Amin Sobh ◽

Charlotte Kaestner ◽

Jianping Li ◽

Alberto Riva ◽

Richard Lynn Bennett ◽

...

Keyword(s):

Multiple Myeloma ◽

Er Stress ◽

Candidate Genes ◽

Cell Lines ◽

Adenylate Kinase ◽

Conflicts Of Interest ◽

Essential Gene ◽

Gene Knockout ◽

A Genome

Background: Multiple myeloma (MM)-associated t (4;14) chromosomal translocation leads to overexpression of NSD2, the histone H3 lysine 36 specific methyltransferase. t(4;14) MM patients have a high risk of relapse and NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance in MM cells. The lack of a specific and potent NSD2 inhibitors mandates finding alternative strategies for treating NSD2-high MM. Aim: This study aims to test the hypothesis that NSD2 overexpression in MM cells generates cellular vulnerabilities that can be therapeutically exploited for treatment of t (4;14) MM. Methods: We conducted a genome wide CRISPR-based loss-of-function genetic screen using the human Brunello library in isogenic NSD2-high (NTKO) and NSD2-low (TKO) KMS-11 derived MM cells to define genes whose loss is selectively detrimental to cells with NSD2 overexpression. The cellular dependency of each identified candidate was then investigated across hundreds of human cell lines using the Cancer Dependency Map portal (www.Depmap.org). Candidate genes were validated using CRISPR-Cas9 gene knockout and shRNA knockdown of individual target genes followed by in vitro competitive growth assays and cell viability assays. Results: Our study revealed multiple candidate genes with increased dependency in NSD2-high cells including the adenine nucleotide regulator Adenylate Kinase 2 (AK2). AK2 catalyzes the reversible conversion of ADP to AMP and ATP and can thus modulates energy balance within the cell. Dependency map analysis showed that AK2 is not a commonly essential gene. The top enriched lineages with AK2 dependency included MM with notable representation of t(4;14)-positive MM cell lines. The increased dependency of NTKO and other t (4;14) MM cells on AK2 was confirmed by in vitro competition assays. Disruption of AK2 in TKO cells had a minimal effect on cellular fitness but the dependency on AK2 was restored upon engineered overexpression of NSD2 in these cells. In addition, NSD2-high cells displayed higher sensitivity to the proteasome inhibitor bortezomib than NSD2-low cells suggesting elevated levels of endoplasmic reticulum (ER) stress in cells overexpressing NSD2. Elevated ER stress necessitates increased levels of ATP to refold proteins and could underlie the increased dependency of NSD2-high cells on AK2. Notably, suppression of AK2 increased bortezomib sensitivity in t (4;14) MM cell lines. Conclusions: Our findings indicate that NSD2 high t(4;14) MM may have a vulnerability due to increased proteostatic stress. Accordingly, AK2 inhibition could be used in combination with proteasome inhibitors to treat MM patients with t (4;14) translocations by inducing the accumulation of lethal levels of unfolded proteins. Disclosures No relevant conflicts of interest to declare.

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A Genome-Wide CRISPR/Cas9 Knockout Screen Identifies BEND3 As a Determinant of Sensitivity to UBA1 Inhibition in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-110406 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1350-1350 ◽

Cited By ~ 1

Author(s):

Samir H Barghout ◽

Neil MacLean ◽

G. Wei Xu ◽

Geethu Thomas ◽

Zachary Blatman ◽

...

Keyword(s):

Er Stress ◽

Signaling Pathways ◽

Enzyme Inhibitor ◽

Enrichment Analysis ◽

Fold Increase ◽

Parp Cleavage ◽

Genome Wide ◽

A Genome

Abstract UBA1 is the major ubiquitin-activating enzyme that initiates the ubiquitylation cascade whereby proteins are tagged with mono- or polyubiquitin to mark them for proteasomal degradation or modify their functions. Despite having equal levels of UBA1 protein, AML cell lines and primary AML cells are more dependent on UBA1 activity compared to normal hematopoietic cells, rendering them more vulnerable to UBA1 inhibition. Recently, we demonstrated that inhibiting UBA1 with the small-molecule inhibitor TAK-243 was selectively cytotoxic to a subset of AML cells and stem cells in vitro and in vivo through a mechanism at least partly dependent on inducing ER stress (Leukemia, 2018). To identify potential determinants of sensitivity/resistance to TAK-243 (Millennium Pharmaceuticals, Takeda) in AML, we conducted a genome-wide CRISPR/Cas9 knockout screen in OCI-AML2 cells followed by selection with cytotoxic TAK-243 concentrations corresponding to the IC90 and IC99. By next-generation sequencing and enrichment analysis, we then identified genes whose knockout renders AML cells resistant to TAK-243. We identified 34 hits in the IC90 and 11 hits in the IC99 arms of the screen (cut off FDR < 0.2). These hits are involved in signaling pathways including transcriptional regulation, histone methylation, ubiquitin conjugation, cell cycle progression, mTOR and NF-κB signaling pathways, consistent with the broad range of pathways regulated by UBA1-mediated ubiquitylation. We focused our investigation on BEN domain-containing protein 3 (BEND3) that ranked as a top hit in both arms (FDR = 0.0012). Compared to control, all 6 BEND3-targeting gRNAs were enriched up to 10,000 times. BEND3 is a transcriptional repressor that regulates heterochromatin organization. To validate the screen results, we independently knocked out BEND3 in OCI-AML2 cells using the 4 top performing gRNAs in the screen. We confirmed target knockout by immunoblotting. BEND3 knockout did not alter the basal proliferation rate of the cells. However, knockout of BEND3 rendered OCI-AML2 cells resistant to TAK-243 with up to a 4-fold increase in the IC50 by the MTS assay. Resistance to TAK-243 was confirmed by Annexin V/PI staining, PARP cleavage, and colony-forming assays. Cells were cross resistant to the NEDD8-activating enzyme inhibitor pevonedistat (2-fold IC50 increase), but not bortezomib or daunorubicin. As assessed by immunoblotting, BEND3 knockout did not change expression of UBA1, or the related enzymes UBA2, UBA3, or UBA6. BEND3 knockout was associated with reduced induction of ER stress as assessed by levels of CHOP and ATF4 after TAK-243 treatment. Conclusions Through a genome-wide CRISPR screen, we identified BEND3 as a determinant of sensitivity to TAK-243 in AML. Mechanistically, lack of BEND3 expression dampens the ER stress response to UBA1 inhibition. Thus, these results may highlight a new mechanism of sensitivity to TAK-243. Disclosures Schimmer: Jazz Pharmaceuticals: Consultancy; Medivir AB: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Antioxidant Effect of Lycium barbarum Leaf through Inflammatory and Endoplasmic Reticulum Stress Mechanism

Antioxidants ◽

10.3390/antiox10010020 ◽

2020 ◽

Vol 10 (1) ◽

pp. 20

Author(s):

So Rok Lee ◽

Mi-Yeong An ◽

Hye-Jeong Hwang ◽

Ju-Gyeong Yoon ◽

Jin Ah Cho

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Genome Wide Association Study ◽

No Production ◽

Dependent Manner ◽

Lycium Barbarum ◽

Genome Wide ◽

A Genome ◽

Anti Inflammatory

Although the prevalence and incidence of inflammatory bowel disease (IBD), a defective immune response of the gastrointestinal tract, has been increasing in North America and Western Europe, recent studies have shown that this disease is also increasing rapidly in Asia. Several studies have been searching for functional foods that can prevent or reduce IBD symptoms because the drug treatments for IBD are expensive with complications. Genome-Wide Association Study (GWAS), an observational study of a genome-wide set of genetic variants in different individuals, showed that endoplasmic reticulum (ER) stress is one of the causes of IBD. Previously, we reported the effects of Lyciumbarbarum fruit and this study investigated the effects of Lycium barbarum leaf (LL) on inflammation and ER stress of the intestine. The paracellular permeability, antioxidant, and anti-inflammatory response were measured on polarized Caco-2 cells. The ER stress pathway and pro-inflammatory cytokines were evaluated on MEF-knockout cell lines, and on the intestines of the mice fed a high-fat diet with lipopolysaccharide-induced inflammation. Our data showed that the LL pretreatment strengthened the tight junction integrity and reduced NO production both in the presence and in the absence of inflammation. Furthermore, LL inhibited ER stress and inflammation via IRE1α and XBP1 in vitro as well as in the inflamed intestines of mice, highlighting the antioxidant and anti-inflammatory function of LL in an IRE1α-XBP1-dependent manner.

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EGFR Expression und der Effekt von Gefitinib auf das in-vitro Wachstum von Tumoren der Gallenwege

Zeitschrift für Gastroenterologie ◽

10.1055/s-2004-816125 ◽

2004 ◽

Vol 42 (01) ◽

Cited By ~ 1

Author(s):

J Feisthammel ◽

M Wiedmann ◽

J Mössner ◽

K Caca

Keyword(s):

Egfr Expression

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STEM-26. SMALL MOLECULE DRUG CBL0137 INHIBITS THE FACT COMPLEX AND SENSITIZES GLIOBLASTOMA CANCER STEM CELLS TO RADIOTHERAPY

Neuro-Oncology ◽

10.1093/neuonc/noaa215.843 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii201-ii202

Author(s):

Miranda Tallman ◽

Abigail Zalenski ◽

Amanda Deighen ◽

Morgan Schrock ◽

Sherry Mortach ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

High Rate ◽

Orthotopic Model ◽

In Vivo Models ◽

Specific Cytotoxicity ◽

Treatment Paradigm ◽

Cancer Agent

Abstract Glioblastoma (GBM) is a malignant brain tumor with nearly universal recurrence. GBM cancer stem cells (CSCs), a subpopulation of radio- and chemo-resistant cancer cells capable of self-renewal, contribute to the high rate of recurrence. The anti-cancer agent, CBL0137, inhibits the FACT (facilitates chromatin transcription) complex leading to cancer cell specific cytotoxicity. Here, we show that CBL0137 sensitized GBM CSCs to radiotherapy using both in vitro and in vivo models. Treatment of CBL0137 combined with radiotherapy led to increased DNA damage in GBM patient specimens and failure to resolve the damage led to decreased cell viability. Using clonogenic assays, we confirmed that CBL0137 radiosensitized the CSCs. To validate that combination therapy impacted CSCs, we used an in vivo subcutaneous model and showed a decrease in the frequency of cancer stem cells present in tumors as well as decreased tumor volume. Using an orthotopic model of GBM, we confirmed that treatment with CBL0137 followed by radiotherapy led to significantly increased survival compared to either treatment alone. Radiotherapy remains a critical component of patient care for GBM, even though there exists a resistant subpopulation. Radio-sensitizing agents, including CBL0137, pose an exciting treatment paradigm to increase the efficacy of irradiation, especially by inclusively targeting CSCs.

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Combination of Niraparib, Cisplatin and Twist Knockdown in Cisplatin-Resistant Ovarian Cancer Cells Potentially Enhances Synthetic Lethality through ER-Stress Mediated Mitochondrial Apoptosis Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22083916 ◽

2021 ◽

Vol 22 (8) ◽

pp. 3916

Author(s):

Entaz Bahar ◽

Ji-Ye Kim ◽

Dong-Chul Kim ◽

Hyun-Soo Kim ◽

Hyonok Yoon

Keyword(s):

Ovarian Cancer ◽

Cell Culture ◽

Cell Death ◽

Er Stress ◽

Cisplatin Resistance ◽

Cross Resistance ◽

Ovarian Cancer Cells ◽

Apoptosis Pathway ◽

Platinum Based Chemotherapy

Poly (ADP-ribose) polymerase 1 inhibitors (PARPi) are used to treat recurrent ovarian cancer (OC) patients due to greater survival benefits and minimal side effects, especially in those patients with complete or partial response to platinum-based chemotherapy. However, acquired resistance of platinum-based chemotherapy leads to the limited efficacy of PARPi monotherapy in most patients. Twist is recognized as a possible oncogene and contributes to acquired cisplatin resistance in OC cells. In this study, we show how Twist knockdown cisplatin-resistant (CisR) OC cells blocked DNA damage response (DDR) to sensitize these cells to a concurrent treatment of cisplatin as a platinum-based chemotherapy agent and niraparib as a PARPi on in vitro two-dimensional (2D) and three-dimensional (3D) cell culture. To investigate the lethality of PARPi and cisplatin on Twist knockdown CisR OC cells, two CisR cell lines (OV90 and SKOV3) were established using step-wise dose escalation method. In addition, in vitro 3D spheroidal cell model was generated using modified hanging drop and hydrogel scaffolds techniques on poly-2-hydroxylethly methacrylate (poly-HEMA) coated plates. Twist expression was strongly correlated with the expression of DDR proteins, PARP1 and XRCC1 and overexpression of both proteins was associated with cisplatin resistance in OC cells. Moreover, combination of cisplatin (Cis) and niraparib (Nira) produced lethality on Twist-knockdown CisR OC cells, according to combination index (CI). We found that Cis alone, Nira alone, or a combination of Cis+Nira therapy increased cell death by suppressing DDR proteins in 2D monolayer cell culture. Notably, the combination of Nira and Cis was considerably effective against 3D-cultures of Twist knockdown CisR OC cells in which Endoplasmic reticulum (ER) stress is upregulated, leading to initiation of mitochondrial-mediated cell death. In addition, immunohistochemically, Cis alone, Nira alone or Cis+Nira showed lower ki-67 (cell proliferative marker) expression and higher cleaved caspase-3 (apoptotic marker) immuno-reactivity. Hence, lethality of PARPi with the combination of Cis on Twist knockdown CisR OC cells may provide an effective way to expand the therapeutic potential to overcome platinum-based chemotherapy resistance and PARPi cross resistance in OC.

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A novel phosphoramide compound, DCZ0805, shows potent anti-myeloma activity via the NF-κB pathway

Cancer Cell International ◽

10.1186/s12935-021-01973-1 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Xuejie Gao ◽

Bo Li ◽

Anqi Ye ◽

Houcai Wang ◽

Yongsheng Xie ◽

...

Keyword(s):

Mouse Model ◽

Tumor Burden ◽

High Rate ◽

Cell Counting ◽

Tumor Xenograft ◽

Luciferase Reporter ◽

Therapeutic Effects ◽

Xenograft Mouse Model

Abstract Background Multiple myeloma (MM) is a highly aggressive and incurable clonal plasma cell disease with a high rate of recurrence. Thus, the development of new therapies is urgently needed. DCZ0805, a novel compound synthesized from osalmide and pterostilbene, has few observed side effects. In the current study, we intend to investigate the therapeutic effects of DCZ0805 in MM cells and elucidate the molecular mechanism underlying its anti-myeloma activity. Methods We used the Cell Counting Kit-8 assay, immunofluorescence staining, cell cycle assessment, apoptosis assay, western blot analysis, dual-luciferase reporter assay and a tumor xenograft mouse model to investigate the effect of DCZ0805 treatment both in vivo and in vitro. Results The results showed that DCZ0805 treatment arrested the cell at the G0/G1 phase and suppressed MM cells survival by inducing apoptosis via extrinsic and intrinsic pathways. DCZ0805 suppressed the NF-κB signaling pathway activation, which may have contributed to the inhibition of cell proliferation. DCZ0805 treatment remarkably reduced the tumor burden in the immunocompromised xenograft mouse model, with no obvious toxicity observed. Conclusion The findings of this study indicate that DCZ0805 can serve as a novel therapeutic agent for the treatment of MM.

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