STK33 Kinase Activity Is Nonessential in KRAS-Dependent Cancer Cells

Babij, Carol; Zhang, Yihong; Kurzeja, Robert J.; Munzli, Anke; Shehabeldin, Amro; Fernando, Manory; Quon, Kim; Kassner, Paul D.; Ruefli-Brasse, Astrid; Watson, Vivienne J.; Fajardo, Flordeliza; Jackson, Angela; Zondlo, James; Sun, Yu; Ellison, Aaron R.; Plewa, Cherylene A.; San, Miguel Tisha; Robinson, John H.; McCarter, John D.; Schwandner, Ralf; Judd, Ted; Carnahan, Josette; Dussault, Isabelle

doi:10.1158/0008-5472.can-11-0778

Cited by 108 publications

(88 citation statements)

References 15 publications

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“…The values for duplicate shRNAs were averaged to generate a single value for each shRNA. The approximately 18 to 19 independent shRNAs/gene were used to estimate a P value for each gene as previously described (18), which was then used to calculate a q-value corrected for multiple hypothesis testing using the method of Hochberg and Benjamini (19).…”

Section: Generation Of Adcmentioning

confidence: 99%

SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm

et al. 2015

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Antibody-drug conjugates (ADC) target cytotoxic drugs to antigen-positive cells for treating cancer. After internalization, ADCs with noncleavable linkers are catabolized to amino acidlinker-warheads within the lysosome, which then enter the cytoplasm by an unknown mechanism. We hypothesized that a lysosomal transporter was responsible for delivering noncleavable ADC catabolites into the cytoplasm. To identify candidate transporters, we performed a phenotypic shRNA screen with an anti-CD70 maytansine-based ADC. This screen revealed the lysosomal membrane protein SLC46A3, the genetic attenuation of which inhibited the potency of multiple noncleavable antibodymaytansine ADCs, including ado-trastuzumab emtansine. In contrast, the potencies of noncleavable ADCs carrying the structurally distinct monomethyl auristatin F were unaffected by SLC46A3 attenuation. Structure-activity experiments suggested that maytansine is a substrate for SLC46A3. Notably, SLC46A3 silencing led to relative increases in catabolite concentrations in the lysosome. Taken together, our results establish SLC46A3 as a direct transporter of maytansine-based catabolites from the lysosome to the cytoplasm, prompting further investigation of SLC46A3 as a predictive response marker in breast cancer specimens. Cancer Res; 75(24); 5329-40. Ó2015 AACR.

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Section: Generation Of Adcmentioning

confidence: 99%

SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm

et al. 2015

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“…Babij and colleagues report that STK33 is dispensable for KRAS mut -dependent cancer cells (1), a finding in contrast to previous investigations (2)(3)(4). We believe the data do not support this conclusion and wish to bring selected methodologic issues to the authors' attention.…”

Section: Stefan Fr€ Ohling and Claudia Schollmentioning

confidence: 59%

STK33 Kinase Is Not Essential in KRAS-Dependent Cells–Letter

Fröhling¹,

Scholl²

2011

Cancer Research

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Babij and colleagues report that STK33 is dispensable for KRAS mut -dependent cancer cells (1), a finding in contrast to previous investigations (2-4). We believe the data do not support this conclusion and wish to bring selected methodologic issues to the authors' attention. Because of space constraints, we have focused on the RNA interference (RNAi) experiments presented.Transient transfection of siRNAs was used to target STK33 and KRAS in leukemia cell lines, but transfection efficiency was not reported. Leukemic cells are difficult to transfect, typically resulting in a large proportion of cells without the respective siRNA whose outgrowth may obscure the effects of gene knockdown, especially when inefficient siRNAs are used.The siRNAs used by Babij and colleagues had inconsistent, context-dependent effects. For example, STK33_7Q, a weak siRNA, also suppressed KRAS and inhibited the growth of some KRAS mut cell lines but not others. Furthermore, while all siRNAs targeting KRAS decreased KRAS mRNA levels, their phenotypic effects in KRAS mut cell lines were highly variable. Surprisingly, these siRNAs had a strong antiproliferative effect in MDA-MB-231, even though this cell line was KRAS-independent in the siRNA screens by Babij and colleagues.There is nominal overlap of two KRAS mut and three KRAS wt cell lines between the siRNA screens conducted by Babij and colleagues and our previous study that used a lentiviral short hairpin RNA (shRNA) library (2). Strikingly, the only two KRAS mut lines included in both studies (PANC-1 and MDA-MB-231) were KRAS-independent in the screens by Babij and colleagues, whereas we observed substantial growth inhibition of PANC-1 and MDA-MB-231 following KRAS knockdown in multiple experimental systems. Given the apparent lack of KRAS essentiality in PANC-1, it is perplexing that these cells were chosen to evaluate the impact of kinase-deficient STK33.To select "hits" from our shRNA screens, we applied relaxed criteria to guard against overlooking genes that, when suppressed, are not "strong killers" but nevertheless segregate with KRAS mut dependence, such as STK33 (2). This strategy was influenced by our experience that the effects of KRAS and STK33 knockdown on adherent cells are weaker in standard tissue culture than in soft agar or immunocompromised mice. Because of the limited sensitivity of cellular assays "on plastic," we confirmed our findings in colony formation and xenotransplantation experiments, a strategy not used by Babij and colleagues. We concur that it is critical to guard against offtarget effects in RNAi studies. This can be achieved by in vitro and in vivo validation of candidate genes. Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

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“…Unfortunately, the dependence of KRAS for STK33 activity was not confirmed in these follow-up studies using RNA interference (RNAi), dominant mutant overexpression, or small-molecule inhibitors of STK33. [54][55][56] Moreover, none of the identified synthetic lethal partners have, to our knowledge, given rise to successful new drug discovery programs to date. Despite the disappointment of the STK33/KRAS synthetic lethality story, it does represent an encouraging, but still all too rare, example of researchers publishing target devalidation data.…”

Section: Synthetic Lethality and Rna Interference Studiesmentioning

confidence: 99%

Addressing the Right Targets in Oncology: Challenges and Alternative Approaches

Stock¹,

Jones²,

Hammonds³

et al. 2015

SLAS Discovery

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Translating existing and emerging knowledge of cancer biology into effective novel therapies remains a great challenge in drug discovery. A firm understanding of the target biology, confidence in the supporting preclinical research, and access to diverse chemical matter is required to lower attrition rates and prosecute targets effectively. Understanding past successes and failures will aid in refining this process to deliver further therapeutic benefit to patients. In this review, we suggest that early oncology drug discovery should focus on selection and prosecution of cancer targets with strong disease biology rather than on more chemically "druggable" targets with only modest disease-linkage. This approach offers higher potential benefit but also increases the need for innovative and alternative approaches. These include using different methods to validate novel targets and identify chemical matter, as well as raising the standards and our interpretation of the scientific literature. The combination of skills required for this emphasizes the need for broader early collaborations between academia and industry.

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STK33 Kinase Activity Is Nonessential in KRAS-Dependent Cancer Cells

Cited by 108 publications

References 15 publications

SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm

SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm

STK33 Kinase Is Not Essential in KRAS-Dependent Cells–Letter

Addressing the Right Targets in Oncology: Challenges and Alternative Approaches

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