The drug efflux pump MDR1 promotes intrinsic and acquired resistance to PROTACs in cancer cells

May combine two claims.

Chronic exposure of A1847 ovarian cancer cells to BBD or CDK9 degraders generated degrader-resistant cells that were more resistant to BBD or CDK9 degraders than parental cells, showing a rightward shift in dose-response cell viability curves.

“The chronically exposed A1847 cells were more resistant to BBD or CDK9 degraders than treatment-naïve (meaning, parental) cells, whereby they showed a rightward shift in dose-response cell viability curves (Fig. 1, B and C, and fig. S1A).”

May combine two claims.

BET protein or CDK9 degrader-resistant cells acquired resistance to paclitaxel, a known substrate of MDR1, as well as were cross-resistant to PROTACs targeting other proteins.

“Notably, BET protein or CDK9 degrader-resistant cells acquired resistance to paclitaxel (fig. S3A), a known substrate of MDR1 (26), as well as were cross-resistant to PROTACs targeting other proteins (fig. S3, B and C).”

May combine two claims.

MDR1 expressing cells required higher concentrations of Thal SNS 032 or dBET6 to reduce target protein levels, with DC50 values overall greater in MDR1-expressing cells compared to MDR1 non-expressing cell lines.

“Notably, MDR1 expressing cells required higher concentrations of Thal SNS 032 (Fig. 4, D to F), or dBET6 (fig. S4, E to G) to reduce target protein levels, with DC50 values overall greater in MDR1-expressing cells compared to MDR1 non-expressing cell lines.”

May combine two claims.

Forced expression of FLAG-MDR1 in SUM159 cells rescued colony formation growth in MZ1-treated cells that could be blocked by tariquidar treatment, signifying overexpression of MDR1 reduces sensitivity towards BET degraders.

“Moreover, forced expression of FLAG-MDR1 in SUM159 cells rescued colony formation growth in MZ1-treated cells that could be blocked by tariquidar treatment, signifying overexpression of MDR1 reduces sensitivity towards BET degraders (Fig. 3, N and O).”

May combine two claims.

FISH analysis of metaphase spreads from dBET6-R cells revealed either many copies of ABCB1 locus localized within three duplicated segments within a very long derivative (7) marker chromosome or many copies of ABCB1 within small extrachromosomal material known as double minutes (dmin), although structures not discernable by Giemsa or DAPI staining, indicating smaller than typical.

“FISH analysis of metaphase spreads from dBET6-R cells revealed either many copies of the ABCB1 locus localized within three duplicated segments within a very long derivative (7) marker chromosome or many copies of the ABCB1 within small extrachromosomal material known as double minutes (dmin) (24), although it is noteworthy that these structures were not discernable by Giemsa or DAPI staining, ind”

May combine two claims.

Nearly all SUM159 parental cells showed normal diploid number for ABCB1 in FISH, whereas drug-resistant SUM159 MZ1-R cells typically had four copies of ABCB1, each residing near a 7-centromere signal, suggesting gain of part or all of two copies of long arm of chromosome 7.

“Nearly all SUM159 parental cells showed a normal diploid number for ABCB1 in FISH analysis, whereas drug-resistant SUM159 MZ1-R cells typically had four copies of ABCB1, each residing near a 7-centromere signal, suggesting gain of part or all of two copies of the long arm of chromosome 7 (Fig. 2I).”

May combine two claims.

Knockdown of IGF2BP3 observed no effect on BRD4 or CDK9 protein levels or induction of apoptosis, suggesting elevated IGF2BP3 does not contribute to degrader activity in resistant cells.

“Additionally, we knocked down IGF2BP3, the other candidate protein commonly upregulated in both dBET6-R and Thal-R cells, and we observed no effect on BRD4 or CDK9 protein levels or induction of apoptosis, suggesting that elevated IGF2BP3 does not contribute to degrader activity in resistant cells (fig. S3D).”

May combine two claims.

Knockdown of ABCB1 reduced cell viability in dBET6-R or Thal-R A1847 cells or MZ1-R SUM159 cells, whereas it exhibited minimal effects in parental cells.

“Knockdown of ABCB1 reduced cell viability in dBET6-R or Thal-R A1847 cells (Fig. 3A) or MZ1-R SUM159 cells (Fig. 3B), whereas it exhibited minimal effects in parental cells.”

May combine two claims.

MDR1 protein levels were upregulated ~3.5-fold in dBET6-R, ~5-5-fold in MZ1-R and ~2.5-fold in Thal-R cells relative to parental cell lines by LFQ analysis.

“MDR1 protein levels were upregulated ~3.5-fold in dBET6-R, ~5–5-fold in MZ1-R and ~2.5-fold in Thal-R cells relative to parental cell lines by LFQ analysis (Fig. 1, L and M, and fig. S1K).”

May combine two claims.

We were unable to generate MZ1-resistant MOLT4 cells and chronic exposure to BET protein degraders did not result in upregulation of ABCB1 mRNA or protein levels.

“In contrast, we were unable to generate MZ1-resistant MOLT4 cells (fig. 3SJ) and chronic exposure to BET protein degraders did not result in upregulation of ABCB1 mRNA or protein levels (Fig. 3, P and Q).”

May combine two claims.

Co-treatment of DLD-1 cells with tariquidar and dBET6 blocked cell viability and colony formation to greater extent than single agent therapies, as well as reduced protein levels of BRD4 target MYC and induced apoptosis.

“Similarly, co-treatment of DLD-1 cells with tariquidar and dBET6 blocked cell viability and colony formation to a greater extent than single agent therapies, as well as reduced protein levels of the BRD4 target MYC and induced apoptosis (Fig. 4, R to T).”

May combine two claims.

Querying TCGA and human protein atlas databases revealed ABCB1 gene expression was frequently altered in tumors from renal, colorectal, and liver cancer patients, whereas elevated MDR1 protein levels were observed in >50% of liver and colorectal cancer specimens by IHC.

“Similarly, querying the cancer genome atlas (TCGA) and human protein atlas databases, revealed that ABCB1 gene expression was frequently altered in tumors from renal, colorectal, and liver cancer patients, whereas elevated MDR1 protein levels were observed in >50% of liver and colorectal cancer specimens by immunohistochemistry (IHC) (31) (fig. S4, A and B).”

May combine two claims.

OVCAR3 and HCT116 cell lines acquired resistance to MZ1 that was accompanied by elevated MDR1 mRNA and protein levels in parental cells, as well as increased sensitivity towards tariquidar-treatments.

“OVCAR3 and HCT116 cell lines acquired resistance to MZ1 (fig. S3, F and G) that was accompanied by elevated MDR1 mRNA and protein levels in parental cells (Fig. 3, P and Q), as well as an increased sensitivity towards tariquidar-treatments (fig. S3, H and I).”

May combine two claims.

Combination therapies involving CDK9 degraders and tariquidar in DLD-1 cells exhibited high drug synergy (Bliss synergy score 45.4) in blocking cell viability in 5-day growth assays and inhibited colony formation over 14-day period better than single agent therapies.

“Combination therapies involving CDK9 degraders and tariquidar in DLD-1 cells exhibited high drug synergy (Bliss synergy score 45.4) in blocking cell viability in 5-day growth assays and inhibited colony formation over a 14-day period better than single agent therapies (Fig. 4, O and P).”

May combine two claims.

Chronic exposure of A1847 cells to BET inhibitor JQ1 did not cause sensitization to tariquidar, suggesting acquired dependency on MDR1 was specific to BBD degrader-resistance.

“Notably, chronic exposure of A1847 cells to BET inhibitor JQ1 did not cause sensitization to tariquidar, suggesting that acquired dependency on MDR1 was specific to BBD degrader-resistance (fig. S3E).”

May combine two claims.

Analysis of ABCB1 mRNA expression across cancer cell line encyclopedia revealed 13% of all cancer cell lines exhibited overexpression of ABCB1, with renal cell carcinomas, colorectal cancer, hepatobiliary (liver) cancer, and neuroblastomas showing greatest frequency of overexpression.

“Analysis of ABCB1 mRNA expression across the cancer cell line encyclopedia (29, 30) revealed that 13% of all cancer cell lines exhibited overexpression of ABCB1, with renal cell carcinomas, colorectal cancer, hepatobiliary (liver) cancer, and neuroblastomas showing the greatest frequency of overexpression (Fig. 4A).”

May combine two claims.

Giemsa-banded metaphase analysis from SUM159 MZ1-R cell line indicated cells were near-triploid and confirmed presence of four copies of 7q, with one copy residing in derivative (7) that appears to contain 7q and all or most of 8q.

“Giemsa-banded metaphase analysis from SUM159 MZ1-R cell line indicated that cells were near-triploid and confirmed the presence of four copies of 7q, with one copy residing in a derivative (7) that appears to contain 7q and all or most of 8q (fig. S2F).”

May combine two claims.

Parental SUM159 cells had overall lower methylation than A1847 cells, and no change or perhaps only minor increase in CpG methylation of ABCB1 promoter was detected in SUM159 MZ1-R cells relative to parental cells.

“Parental SUM159 cells had overall lower methylation than A1847 cells, and no change or perhaps only a minor increase in CpG methylation of the ABCB1 promoter was detected in SUM159 MZ1-R cells relative to parental cells (Fig. 2K).”

May combine two claims.

Degrader-resistant A1847 cells showed amplification of ABCB1, with mean ~12 ABCB1 signals/nucleus in Thal-R cells, and mean ~30 signals/nucleus in dBET6-R cells, whereas only 3 to 4 signals per nucleus were observed for 7 centromere probe in each degrader-resistant cell line.

“Degrader-resistant A1847 cells showed amplification of ABCB1, with a mean of ~12 ABCB1 signals/nucleus in Thal-R cells, and a mean of ~30 signals/nucleus in dBET6-R cells, whereas only 3 to 4 signals per nucleus were observed for 7 centromere probe in each degrader-resistant cell line (Fig. 2H and fig. S2C).”

May combine two claims.

Co-administration of Thal SNS 032 and tariquidar improved protein degradation of CDK9, reduced phosphorylation of Pol II (Ser2) and induced apoptosis uniquely in combination.

“Moreover, co-administration of Thal SNS 032 and tariquidar improved protein degradation of CDK9, reduced phosphorylation of Pol II (Ser2) and induced apoptosis uniquely in the combination (Fig. 4Q).”

May combine two claims.

Ubiquitin ligases were not amongst top upregulated proteins in dBET6-R or Thal-R cells, ruling out a compensatory ligase response.

“Notably, ubiquitin ligases were not amongst the top upregulated proteins in dBET6-R or Thal-R cells, ruling out a compensatory ligase response (data file S1).”

May combine two claims.

FISH analysis of metaphase spreads from Thal-R cell showed a 7q deletion and a long derivative (7) containing a putative homogeneously staining region (hsr) hybridized to ABCB1 probe.

“Notably, FISH analysis of metaphase spreads from Thal-R cell showed a 7q deletion and a long derivative (7) containing a putative homogeneously staining region (hsr) hybridized to the ABCB1 probe (red mid-size arrow) (fig. S2D).”

May combine two claims.

Elevated ABCB1 mRNA and protein levels were confirmed in degrader-resistant A1847 and SUM159 cells by RT-PCR, immunoblot and immunofluorescence, where MDR1 protein was detected at membrane of degrader-resistant cells.

“Elevated ABCB1 mRNA and protein levels were confirmed in degrader-resistant A1847 and SUM159 cells by reverse transcription-polymerase chain reaction (RT-PCR) (Fig. 2A and fig. S2A), immunoblot (Fig. 2B and fig. S2B) and immunofluorescence (Fig. 2, C to E), where MDR1 protein was detected at the membrane of degrader-resistant cells.”

May combine two claims.

Genetic depletion of ABCB1 restored degradation of BET proteins or CDK9 in degrader-resistant cells but not parental cells, re-sensitizing cells to degraders and causing apoptosis.

“Consistent with this, genetic depletion of ABCB1 restored degradation of BET proteins or CDK9 in degrader-resistant cells but not parental cells, re-sensitizing cells to the degraders and causing apoptosis (Fig. 3, C and D).”

May combine two claims.

Reduced protein levels of BRD2, BRD3 and BRD4 were detected in dBET6-R or MZ1-R cells relative to parental cells, suggesting some target degradation was still occurring in these chronically exposed cells.

“Of note, reduced protein levels of BRD2, BRD3 and BRD4 were detected in dBET6-R (Fig. 3C) or MZ1-R (Fig. 3D) cells relative to parental cells, suggesting that some target degradation was still occurring in these chronically exposed cells.”

May combine two claims.

No change in MDR1 levels up to 72 hours after treatment; increased MDR1 protein levels at 7 days post treatment with BBD or CDK9 degraders in A1847 and SUM159 cells.

“We observed no change in MDR1 levels up to 72 hours after treatment; however, we did detect increased MDR1 protein levels at 7 days post treatment with BBD or CDK9 degraders in A1847 and SUM159 cells (Fig. 2, L and M).”

May combine two claims.

RAD001 or lapatinib treatment also sensitized DLD-1 cells to Thal SNS 032, improving degradation of CDK9, and enhancing growth inhibition of colonies.

“RAD001 or lapatinib treatment also sensitized DLD-1 cells to Thal SNS 032, improving the degradation of CDK9 (Fig. 5, P and Q), and enhancing growth inhibition of colonies (Fig. 5R).”

May combine two claims.

The FDA-approved mTOR inhibitor RAD001 can block MDR1 activity and overcome MDR1-mediated drug resistance in cancer cells.

“Here, we showed that the FDA-approved mTOR inhibitor, RAD001, could be used to block MDR1 activity, to overcome MDR1-mediated drug resistance in cancer cells.”

May combine two claims.

SUM159 MZ1-R cells did not show amplification of ABCB1 nor changes in promoter methylation.

“Unlike A1847 degrader-resistant cells, our characterization of SUM159 MZ1-R cells did not show amplification of ABCB1 nor changes in promoter methylation”

May combine two claims.

Co-treatment of DLD-1 cells with KU-0063694 and dBET6 did not improve growth inhibition observed with RAD001 and dBET6 combination, where single agent KU-0063694 treatment completely repressed colony formation.

“Similarly, co-treatment of DLD-1 cells with KU-0063694 and dBET6 did not improve growth inhibition observed with the RAD001 and dBET6 combination, where single agent KU-0063694 treatment completely repressed colony formation.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Resistance to PROTACs in cultured cells has been shown to involve genomic alterations in their E3 ligase targets, such as decreased expression of CRBN, VHL, or CUL2.

“Resistance to PROTACs in cultured cells has been shown to involve genomic alterations in their E3 ligase targets, such as decreased expression of Cereblon (CRBN), Von Hippel Lindau (VHL), or Cullin2 (CUL2) (9–11).”

• cites [9] Ottis P, Palladino C, Thienger P, Britschgi A, Heichinger C, Berrera M, Julien-Laferriere A, Roudnicky F, Kam-Thong T, Bischoff JR, Martoglio B, Pettazzoni P, Cellular Resistance Mechanisms to Targeted Protein Degradation Converge Toward Impairment of the Engaged Ubiquitin Transfer Pathway. ACS Chem. Biol. 14, 2215– 2223 (2019). 31553577Not checked yet

  Not checked in the free run.

• cites [10] Shirasaki R, Matthews GM, Gandolfi S, de Matos Simoes R, Buckley DL, Raja Vora J, Sievers QL, Brüggenthies JB, Dashevsky O, Poarch H, Tang H, Bariteau MA, Sheffer M, Hu Y, Downey-Kopyscinski SL, Hengeveld PJ, Glassner BJ, Dhimolea E, Ott CJ, Zhang T, Kwiatkowski NP, Laubach JP, Schlossman RL, Richardson PG, Culhane AC, Groen RWJ, Fischer ES, Vazquez F, Tsherniak A, Hahn WC, Levy J, Auclair D, Licht JD, Keats JJ, Boise LH, Ebert BL, Bradner JE, Gray NS, Mitsiades CS, Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degra sourceNot checked yet

  Not checked in the free run.

• cites [11] Zhang L, Riley-Gillis B, Vijay P, Shen Y, Acquired Resistance to BET-PROTACs(Proteolysis Targeting Chimeras) Caused by Genomic Alterations in Core Components of E3 ligase Complexes. Mol. Cancer Ther, molcanther.1129.2018 (2019). 31064868 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

PROTACs are small molecules with two functional ends, one binding the protein of interest and the other binding an E3 ubiquitin ligase, bringing the ligase to the target protein, leading to its ubiquitination and degradation by the proteasome.

“PROTACs are small molecules with two functional ends, wherein one end binds to the protein of interest while the other binds to an E3 ubiquitin ligase (2, 3), bringing the ubiquitin ligase to the target protein, leading to its ubiquitination and subsequent degradation by the proteasome.”

• cites [2] Bondeson DP, Smith BE, Burslem GM, Buhimschi AD, Hines J, Jaime-Figueroa S, Wang J, Hamman BD, Ishchenko A, Crews CM, Lessons in PROTAC Design from Selective Degradation with a Promiscuous Warhead. Cell Chemical Biology 25, 78– 87.e75 (2018). 29129718 PMC5777153 sourceNot checked yet

  Not checked in the free run.

• cites [3] Lai AC, Crews CM, Induced protein degradation: an emerging drug discovery paradigm. Nat Rev Drug Discov 16, 101– 114 (2017). 27885283 PMC5684876 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Upregulation of MDR1 has been shown to convey drug resistance to many anti-cancer drugs including chemotherapy agents, kinase inhibitors, and other targeted agents.

“Upregulation of the drug efflux pump encoded by ABCB1—MDR1 (multidrug resistance 1), a member of the superfamily of ATP-binding cassette (ABC) transporters—has been shown to convey drug resistance to many anti-cancer drugs including chemotherapy agents, kinase inhibitors, and other targeted agents (12).”

• cites [12] Fletcher JI, Williams RT, Henderson MJ, Norris MD, Haber M, ABC transporters as mediators of drug resistance and contributors to cancer cell biology. Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy 26, 1– 9 (2016). 27180306 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Degrader-resistant cells acquired dependency on MDR1-dependent PROTAC efflux, which permitted cells to survive in continued presence of BBD or CDK9 degraders.

“Together, these findings demonstrate that degrader-resistant cells acquired dependency on MDR1-dependent PROTAC efflux, which permitted cells to survive in the continued presence of BBD or CDK9 degraders.”

May combine two claims.

Knockdown of ABCB1 resulted in reduction of BBD protein levels and increase in cleavage of PARP, signifying degrader-resistant cells were dependent on ABCB1 to maintain residual BRD2, BRD3 and BRD4 protein levels for survival.

“However, knockdown of ABCB1 resulted in a reduction of BBD protein levels and an increase in cleavage of PARP, signifying that the degrader-resistant cells were dependent on ABCB1 to maintain the residual BRD2, BRD3 and BRD4 protein levels for survival.”

May combine two claims.

Continuous exposure of A1847 cells to BBD or CDK9 degraders reduced target degradation efficiency, requiring increased concentrations of PROTACs to achieve DC50 observed in parental cells.

“Together, these findings demonstrated continuous exposure of A1847 cells to BBD or CDK9 degraders reduced target degradation efficiency, thus requiring increased concentrations of PROTACs to achieve DC50 observed in parental cells.”

May combine two claims.

Lapatinib is an FDA approved drug for treatment of several HER2 driven cancers and has been shown to also directly inhibit drug efflux activity of MDR1, both in cancer cells and in in vivo tumor models.

“The ErbB receptor inhibitor lapatinib is an U.S. Food and Drug Administration (FDA) approved drug for the treatment of several HER2 driven cancers and has been shown to also directly inhibit the drug efflux activity of MDR1, both in cancer cells and in in vivo tumor models (36).”

• cites [36] Dai C.-l., Tiwari AK, Wu C-P, Su X-D, Wang S-R, Liu D.-g., Ashby CR, Huang Y, Robey RW, Liang Y.-j., Chen L.-m., Shi C-J, Ambudkar SV, Chen Z-S, Fu L.-w., Lapatinib (Tykerb, GW572016) reverses multidrug resistance in cancer cells by inhibiting the activity of ATP-binding cassette subfamily B member 1 and G member 2. Cancer Res. 68, 7905– 7914 (2008). 18829547 PMC2652245 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

No colonies resistant to BBD-tariquidar treatments over 14-day period, suggesting co-targeting MDR1 with BBD degraders provides durable growth inhibition for at least two weeks.

“Notably, we did not observe colonies resistant to the BBD-tariquidar treatments over the 14-day period, suggesting that co-targeting MDR1 with BBD degraders provides durable growth inhibition for at least two weeks.”

Classified as probably not a substantial claim.

Elevated levels of MDR1 have been shown to promote drug resistance in cancer cells via efflux of large hydrophobic molecules, such as chemotherapy agents.

“Elevated levels of MDR1 have been shown to promote drug resistance in cancer cells via efflux of large hydrophobic molecules, such as chemotherapy agents (26).”

• cites [26] Vaidyanathan A, Sawers L, Gannon A-L, Chakravarty P, Scott AL, Bray SE, Ferguson MJ, Smith G, ABCB1 (MDR1) induction defines a common resistance mechanism in paclitaxel- and olaparib-resistant ovarian cancer cells. Br. J. Cancer 115, 431– 441 (2016). 27415012 PMC4985349 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Co-treatment of DLD-1 cells with FAK degrader or MEK1/2 degrader and tariquidar enhanced protein reduction relative to single agent therapies, suggesting overexpression of MDR1 can promote resistance to degrader therapies, independent of protein target.

“Additionally, co-treatment of DLD-1 cells with FAK degrader (FAK-degrader-1) (32) or MEK1/2 degrader (MS432) (33) and tariquidar enhanced the protein reduction relative to single agent therapies (fig. S4, J and K), suggesting overexpression of MDR1 can promote resistance to degrader therapies, independent of protein target.”

May combine two claims.

Withdrawal of PROTACs from degrader-resistant cell lines for 2 or 7 days did not impact MDR1 protein levels, suggesting upregulation of MDR1 was an adaptive but stable response.

“Notably, withdrawal of the PROTACs from the degrader-resistant cell lines for 2 or 7-days did not impact MDR1 protein levels, suggesting that the upregulation of MDR1 was an adaptive but stable response (Fig. 2, Q and R).”

May combine two claims.

Levels of MDR1 did not appear to further increase in A1847 cells from 7-day time point over 30-day period, suggesting maximal upregulation of MDR1 occurred ~7 days post-treatment with dBET6 or Thal SNS 032.

“The levels of MDR1 did not appear to further increase in A1847 cells from the 7-day time point over the 30-day period, suggesting maximal upregulation of MDR1 occurred ~7 days post-treatment with dBET6 or Thal SNS 032 (Fig. 2, N and O).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Hypomethylation of ABCB1 promoter has been shown to promote drug resistance to chemotherapies through increased ABCB1 expression and drug pump activity.

“Hypomethylation of the ABCB1 promoter has been shown to promote drug resistance to chemotherapies through increased ABCB1 expression and drug pump activity (25).”

• cites [25] Reed K, Hembruff SL, Sprowl JA, Parissenti AM, The temporal relationship between ABCB1 promoter hypomethylation, ABCB1 expression and acquisition of drug resistance. The Pharmacogenomics Journal 10, 489– 504 (2010). 20125118 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Mechanism of ABCB1 upregulation could be attributed to gene amplification and/or reduced CpG promoter methylation in A1847 cells.

“The mechanism of ABCB1 upregulation could be attributed to gene amplification and/or reduced CpG promoter methylation in A1847 cells.”

May combine two claims.

Not all cancer cells will upregulate MDR1 following continuous degrader exposure; in our studies, 4 out of 5 cancer cell lines induced MDR1 causing degrader-resistance.

“These findings suggest that not all cancer cells will upregulate MDR1 following continuous degrader exposure; in our studies, 4 out of 5 cancer cell lines induced MDR1 causing degrader-resistance.”

May combine two claims.

Reductions in promoter methylation of ABCB1 were contributing to increased ABCB1 expression observed in A1847 dBET6-R and Thal-R cells but not in SUM159 MZ1-R cells.

“Together, these findings suggested reductions in promoter methylation of ABCB1 were contributing to the increased ABCB1 expression observed in A1847 dBET6-R and Thal-R cells but not in SUM159 MZ1-R cells.”

May combine two claims.

Level of MDR1 gradually increased in response to MZ1-treatment over 30-day period in SUM159 cells, indicating longer period required for SUM159 to upregulate maximal MDR1 compared to A1847 cells.

“In contrast, the level of MDR1 gradually increased in response to MZ1-treatment over the 30-day period in SUM159 cells (Fig. 2P), indicating that a longer period of time is required for the SUM159 cells to upregulate the maximal level of MDR1 protein compared to A1847 cells.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Amplification of ABCB1 has been previously shown to occur in chemotherapy-resistant cells leading to increased MDR1 drug efflux activity.

“Amplification of ABCB1 has been previously shown to occur in chemotherapy-resistant cells leading to increased MDR1 drug efflux activity (23).”

• cites [23] Genovese I, Ilari A, Assaraf YG, Fazi F, Colotti G, Not only P-glycoprotein: Amplification of the ABCB1-containing chromosome region 7q21 confers multidrug resistance upon cancer cells by coordinated overexpression of an assortment of resistance-related proteins. Drug Resistance Updates 32, 23– 46 (2017). 29145976 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Differences in proteome response could be attributed to distinct recruitment ligases used by MZ1 (VHL) or dBET6 (CRBN), as well as established differences in selectivity for BBDs, where MZ1 preferentially degrades BRD4, whereas dBET6 degrades BRD2, BRD3 and BRD4 equivalently.

“These differences could be attributed to the distinct recruitment ligases used by MZ1 (VHL) or dBET6 (CRBN), as well as the established differences in selectivity for the BBDs, where MZ1 preferentially degrades BRD4 (17), whereas dBET6 degrades BRD2, BRD3 and BRD4, equivalently (16).”

• cites [17] Zengerle M, Chan K-H, Ciulli A, Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4. ACS Chem. Biol. 10, 1770– 1777 (2015). 26035625 PMC4548256 sourceNot checked yet

  Not checked in the free run.

• cites [16] Winter GE, Mayer A, Buckley DL, Erb MA, Roderick JE, Vittori S, Reyes JM, di Iulio J, Souza A, Ott CJ, Roberts JM, Zeid R, Scott TG, Paulk J, Lachance K, Olson CM, Dastjerdi S, Bauer S, Lin CY, Gray NS, Kelliher MA, Churchman LS, Bradner JE, BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment. Mol. Cell 67, 5– 18.e19 (2017). 28673542 PMC5663500 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Mice were randomly split into treatment arms.

“Mice were randomly split into the following treatment arms: vehicle, MS934 (50 mg/kg), Lapatinib (100 mg/kg) or the combination (using the same dose as monotherapies)”

Classified as probably not a substantial claim.

Statistical significance was assessed using Student's t-tests with P values ≤ 0.05 considered significant.

“Student’s t-tests were performed for statistical analyses and P values ≤ 0.05 were considered significant.”

• Also stated in: § Growth Assays · plain

  “Student’s t-tests were performed for statistical analyses and p values ≤ 0.05 were considered significant.”

• Also stated in: § qRT-PCR · plain

  “Student’s t-tests were performed for statistical analyses and P values ≤ 0.05 were considered significant.”

Classified as probably not a substantial claim.

Combination therapies of lapatinib and either KRASG12C or MEK inhibitors provided more durable therapies in tumor models.

“whereby combination therapies of lapatinib and either KRASG12C or MEK inhibitors provided more durable therapies in tumor models (40, 58).”

• cites [40] Sun C, Hobor S, Bertotti A, Zecchin D, Huang S, Galimi F, Cottino F, Prahallad A, Grernrum W, Tzani A, Schlicker A, Wessels Lodewyk F. A., Smit Egbert F., Thunnissen E, Halonen P, Lieftink C, Beijersbergen Roderick L., Di Nicolantonio F, Bardelli A, Trusolino L, Bernards R, Intrinsic Resistance to MEK Inhibition in KRAS Mutant Lung and Colon Cancer through Transcriptional Induction of ERBB3. Cell Reports 7, 86– 93 (2014). 24685132 sourceNot checked yet

  Not checked in the free run.

• cites [58] Uribe ML, Marrocco I, Yarden Y, EGFR in Cancer: Signaling Mechanisms, Drugs, and Acquired Resistance. Cancers (Basel) 13, (2021). PMC8197917 34206026 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

ErbB receptors are frequently altered in cancers.

“ErbB receptors are frequently altered in cancers”

• cites [58] Uribe ML, Marrocco I, Yarden Y, EGFR in Cancer: Signaling Mechanisms, Drugs, and Acquired Resistance. Cancers (Basel) 13, (2021). PMC8197917 34206026 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

KRAS mutations occur in nearly 40% of colorectal cancer patients, supporting therapies that target KRAS effectors such as MEK-ERK signaling pathway.

“KRAS mutations occur in nearly 40% of colorectal cancer (CRC) patients, supporting therapies that target KRAS effectors such as the MEK-ERK signaling pathway (38).”

• cites [38] The N Cancer Genome Atlas, Comprehensive Molecular Characterization of Human Colon and Rectal Cancer. Nature 487, 330– 337 (2012). 22810696 PMC3401966 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Investigators were not blinded to group identity.

“Investigators were not blinded to group identity.”

May combine two claims.

Treatment of degrader-resistant cell lines with RAD001 or lapatinib resulted in apoptosis similar to tariquidar treatment, demonstrating RAD001 or lapatinib can inhibit MDR1 function overcoming MDR1-driven degrader-resistance.

“Moreover, treatment of degrader-resistant cell lines with RAD001 or lapatinib resulted in apoptosis similar to tariquidar treatment (Fig. 5J), demonstrating RAD001 or lapatinib can inhibit MDR1 function overcoming MDR1-driven degrader-resistance.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Samples in the reported experiments were run in biological triplicates (N=3).

“Samples were run in biological triplicates (N=3)”

• Also stated in: § Growth Assays · plain

  “Samples were run in biological triplicates (N=3)”

• Also stated in: § Growth Assays · plain

  “Samples were run in biological triplicates (N=3)”

Classified as probably not a substantial claim.

Recently, MEK1/2 degraders have been developed that show potent anti-growth properties in RAS-RAF altered cancers.

“Recently, MEK1/2 degraders have been developed that show potent anti-growth properties in RAS-RAF altered cancers (33).”

• cites [33] Hu J, Wei J, Yim H, Wang L, Xie L, Jin MS, Kabir M, Qin L, Chen X, Liu J, Jin J, Potent and Selective Mitogen-Activated Protein Kinase Kinase 1/2 (MEK1/2) Heterobifunctional Small-molecule Degraders. J. Med. Chem. 63, 15883– 15905 (2020). 33284613 PMC7770057 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Regulation of ABCB1 expression in cancer cells is complex, with multiple mechanisms functioning cooperatively to increase ABCB1 levels in drug resistance, including gene amplification and/or promoter hypomethylation.

“The regulation of ABCB1 expression in cancer cells is complex, with multiple mechanisms functioning cooperatively to increase ABCB1 levels in drug resistance, including gene amplification and/or promoter hypomethylation (23, 25, 47).”

• cites [23] Genovese I, Ilari A, Assaraf YG, Fazi F, Colotti G, Not only P-glycoprotein: Amplification of the ABCB1-containing chromosome region 7q21 confers multidrug resistance upon cancer cells by coordinated overexpression of an assortment of resistance-related proteins. Drug Resistance Updates 32, 23– 46 (2017). 29145976 sourceNot checked yet

  Not checked in the free run.

• cites [25] Reed K, Hembruff SL, Sprowl JA, Parissenti AM, The temporal relationship between ABCB1 promoter hypomethylation, ABCB1 expression and acquisition of drug resistance. The Pharmacogenomics Journal 10, 489– 504 (2010). 20125118 sourceNot checked yet

  Not checked in the free run.

• cites [47] Chen KG, Sikic BI, Molecular pathways: regulation and therapeutic implications of multidrug resistance. Clinical cancer research: an official journal of the American Association for Cancer Research 18, 1863– 1869 (2012). 22344233 PMC3359695 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Increased PI3K/AKT and/or Raf signaling has been associated with upregulation of ABCB1 in drug resistance.

“Notably, increased PI3K/AKT and/or Raf signaling has been associated with upregulation of ABCB1 in drug resistance (47).”

• cites [47] Chen KG, Sikic BI, Molecular pathways: regulation and therapeutic implications of multidrug resistance. Clinical cancer research: an official journal of the American Association for Cancer Research 18, 1863– 1869 (2012). 22344233 PMC3359695 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

ErbB receptor signaling promotes resistance to a variety of target agents including pan-Tyrosine Kinases (TK), AKT, RAF, MEK, and ERK inhibitors.

“ErbB receptor signaling has been shown to promote resistance to a variety of target agents including pan-Tyrosine Kinases (TK), AKT, RAF, MEK, and ERK inhibitors (60)”

• cites [60] Arteaga CL, Engelman JA, ERBB receptors: from oncogene discovery to basic science to mechanism-based cancer therapeutics. Cancer Cell 25, 282– 303 (2014). 24651011 PMC4018830 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Animal studies were conducted in accordance with Institutional Animal Care and Use Committee guidelines (Fox Chase Cancer Center IACUC #16-16).

“Animal studies were conducted in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee (Fox Chase Cancer Center institutional animal care and use committee (IACUC) # 16–16).”

Probably not a substantial claim: may not pass S3 (load-bearing).

No MDR1 inhibitors are FDA-approved for cancer therapy.

“no MDR1 inhibitors FDA-approved for cancer therapy (34).”

• cites [34] Lai J-I, Tseng Y-J, Chen M-H, Huang C-YF, Chang PM-H, Clinical Perspective of FDA Approved Drugs With P-Glycoprotein Inhibition Activities for Potential Cancer Therapeutics. Front. Oncol. 10, 2336 (2020). PMC7704056 33312947 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Resistance to MEK inhibitors in KRAS-mutant colorectal cancer cells is mediated by activation of ErbB receptors and downstream RAF-MEK-ERK and PI3K/AKT signaling.

“Moreover, resistance to MEK inhibitors in KRAS-mutant colorectal cancer cells is mediated by activation of ErbB receptors and downstream RAF-MEK-ERK and PI3K/AKT signaling (40).”

• cites [40] Sun C, Hobor S, Bertotti A, Zecchin D, Huang S, Galimi F, Cottino F, Prahallad A, Grernrum W, Tzani A, Schlicker A, Wessels Lodewyk F. A., Smit Egbert F., Thunnissen E, Halonen P, Lieftink C, Beijersbergen Roderick L., Di Nicolantonio F, Bardelli A, Trusolino L, Bernards R, Intrinsic Resistance to MEK Inhibition in KRAS Mutant Lung and Colon Cancer through Transcriptional Induction of ERBB3. Cell Reports 7, 86– 93 (2014). 24685132 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Small molecule inhibitors of MDR1 have shown limited therapeutic benefit in clinical trials.

“these drugs have shown limited therapeutic benefit”

• cites [34] Lai J-I, Tseng Y-J, Chen M-H, Huang C-YF, Chang PM-H, Clinical Perspective of FDA Approved Drugs With P-Glycoprotein Inhibition Activities for Potential Cancer Therapeutics. Front. Oncol. 10, 2336 (2020). PMC7704056 33312947 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Cell lines used were verified by IDEXX laboratories and free of mycoplasma.

“Cell lines were verified by IDEXX laboratories and free of mycoplasma.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Images presented in Figure 2 are representative of biological triplicates analyzed.

“Images presented in Figure 2 are representative of biological triplicates analyzed.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Renal cell carcinoma exhibits frequent overexpression of MDR1.

“which exhibits frequent overexpression of MDR1 (55).”

• cites [55] Walsh N, Larkin A, Kennedy S, Connolly L, Ballot J, Ooi W, Gullo G, Crown J, Clynes M, O’Driscoll L, Expression of multidrug resistance markers ABCB1 (MDR-1/P-gp) and ABCC1 (MRP-1) in renal cell carcinoma. BMC Urol. 9, 6 (2009). 19552816 PMC2723136 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

RAD001 is currently used to treat several cancers, including renal cell carcinoma (RCC).

“RAD001 is currently used to treat several cancers (53), including renal cell carcinoma (RCC) (54)”

• cites [53] Murugan AK, mTOR: Role in cancer, metastasis and drug resistance. Semin. Cancer Biol. 59, 92– 111 (2019). 31408724 sourceNot checked yet

  Not checked in the free run.

• cites [54] Faes S, Demartines N, Dormond O, Mechanistic Target of Rapamycin Inhibitors in Renal Cell Carcinoma: Potential, Limitations, and Perspectives. Frontiers in Cell and Developmental Biology 9, 459 (2021). PMC8005589 33791295 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

With 5 animals per group, the study had 80% power to detect a difference of 0.34 in rates of tumor growth between any two groups at the 0.05 significance level, assuming unit standard deviation for log tumor volume and correlation 0.5 for serial measurements.

“With 5 animals per group, we were able to detect a difference of 0.34 in the rates of tumor growth (between any two groups) and achieve 80% power at the 0.05 significance level. This is based on unit standard deviation for the logarithm of tumor volume and assumes a correlation of 0.5 for serial measurements for each mouse.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Lapatinib is a highly selective EGFR, ERBB2 and ERBB4 inhibitor that is currently FDA-approved for the treatment of a variety of malignancies.

“Lapatinib is a highly selective EGFR, ERBB2 and ERBB4 inhibitor that is currently FDA-approved for the treatment of a variety of malignancies (59).”

• cites [59] Opdam FL, Guchelaar H-J, Beijnen JH, Schellens JHM, Lapatinib for advanced or metastatic breast cancer. The oncologist 17, 536– 542 (2012). 22477724 PMC3336826 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Activation of ErbB receptors promotes resistance to KRASG12C or MEK inhibitors in colorectal cancer.

“Activation of ErbB receptors has been shown to promote resistance to KRASG12C or MEK inhibitors in CRC”

• cites [40] Sun C, Hobor S, Bertotti A, Zecchin D, Huang S, Galimi F, Cottino F, Prahallad A, Grernrum W, Tzani A, Schlicker A, Wessels Lodewyk F. A., Smit Egbert F., Thunnissen E, Halonen P, Lieftink C, Beijersbergen Roderick L., Di Nicolantonio F, Bardelli A, Trusolino L, Bernards R, Intrinsic Resistance to MEK Inhibition in KRAS Mutant Lung and Colon Cancer through Transcriptional Induction of ERBB3. Cell Reports 7, 86– 93 (2014). 24685132 sourceNot checked yet

  Not checked in the free run.

• cites [58] Uribe ML, Marrocco I, Yarden Y, EGFR in Cancer: Signaling Mechanisms, Drugs, and Acquired Resistance. Cancers (Basel) 13, (2021). PMC8197917 34206026 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Epigenetic components, chromatin remodeling, transcriptional factors and survival signaling can contribute to the regulation of ABCB1 expression in cancer cells.

“Studies have shown epigenetic components, chromatin remodeling (48), transcriptional factors (49) and survival signaling (50) all can contribute to the regulation of ABCB1 expression in cancer cells.”

• cites [48] Baker EK, Johnstone RW, Zalcberg JR, El-Osta A, Epigenetic changes to the MDR1 locus in response to chemotherapeutic drugs. Oncogene 24, 8061– 8075 (2005). 16091741 sourceNot checked yet

  Not checked in the free run.

• cites [49] Chen KG, Sale S, Tan T, Ermoian RP, Sikic BI, CCAAT/enhancer-binding protein beta (nuclear factor for interleukin 6) transactivates the human MDR1 gene by interaction with an inverted CCAAT box in human cancer cells. Mol. Pharmacol. 65, 906 (2004). 15044620 sourceNot checked yet

  Not checked in the free run.

• cites [50] Liu R, Chen Y, Liu G, Li C, Song Y, Cao Z, Li W, Hu J, Lu C, Liu Y, PI3K/AKT pathway as a key link modulates the multidrug resistance of cancers. Cell Death Dis. 11, 797 (2020). 32973135 PMC7515865 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Upregulation of the MDR1 drug pump is a common mechanism of resistance to several chemotherapies.

“Upregulation of the MDR1 drug pump has been reported as a common mechanism of resistance to several chemotherapies (26).”

• cites [26] Vaidyanathan A, Sawers L, Gannon A-L, Chakravarty P, Scott AL, Bray SE, Ferguson MJ, Smith G, ABCB1 (MDR1) induction defines a common resistance mechanism in paclitaxel- and olaparib-resistant ovarian cancer cells. Br. J. Cancer 115, 431– 441 (2016). 27415012 PMC4985349 sourceNot checked yet

  Not checked in the free run.