Cancer Therapeutics

The promise of “precision oncology” relies on decoding the molecular signatures of tumors to make predictions about effective therapies. The prevailing wisdom is that precision therapies will arise from identifying and targeting "drivers" of oncogenic transformation (e.g., mutated oncogenes). However, this approach has met with limited clinical success, particularly for some of the most devastating and difficult to treat cancers. Glioblastoma multiforme (GBM) is the most aggressive and common form of brain cancer in adults: approximately 90% of GBM patients die within two years of diagnosis with current standard of care therapy. To identify novel GBM therapeutic targets, we have developed a paradigm that focuses not on targeting oncogenic drivers, but instead on normal cellular pathways that are sensitized uniquely in GBM cells and not their healthy counterparts. We have isolated patient-derived GBM “stem cells” (GSCs) and compared them with non-GBM neuronal progenitor cells (NPCs). Every gene is then systematically inactivated in each set of samples to identify genes essential for the self-renewal and growth of only the GSCs. As a result, we have now identified 100s of candidate targets, which are essential to GSCs but which have reduced requirement in NPCs and other normal cells.

Published candidate therapeutic targets:

Knockdown of BUB1B "wrecks" mitosis of GBM stem cells but not NSCs

BubR1-GLEBs domain. BubR1 is a multiple functional protein that plays key roles implicated in mitotic checkpoint control, mitotic timing, and regulating kinetochore-microtubule attachment. We found that certain GBM isolates are sensitive to inhibition of BubR1's GLE2p-binding sequence (GLEBS) domain. We have shown, for example, that in BubR1 knockout cells that BubR1's GLEBs domain becomes essential for kinetochore-microtubule attachment and viability only after oncogenic activation of the RTK/Ras-pathway (Ding et al., Cancer Discovery, 2013). Importantly, we were able to find a key molecular indicator on the kinetochore that predicts whether GBM or other cell types will be sensitive to loss of BubR1's GLEBs domain function. We are currently working with Dr. Jim Olson's group (Clinical Research Division) to identify molecules that will interfere with BubR1's GLEBs domain activity.

Model for BuGZ function

BuGZ-GLEBs domain. BuGZ was isolated from an RNAi screen targeting putative human transcription factors to identify key regulators of GSC expansion. Its official name is ZNF207, a previously uncharacterized C2–H2 zinc-finger domain gene and putative transcription factor. We renamed the gene BuGZ (Bub3 interacting GLEBS and Zinc finger domain containing protein) and demonstrated that BuGZ is a novel kinetochore component that binds to and stabilizes Bub3 during interphase and mitosis. Just like BubR1, BuGZ binds to Bub3 through a highly conserved GLEBS domain. As with BubR1, we found the cancer-specific requirement for BuGZ was limited to its GLEBS domain, which mediates BuGZ kinetochore localization (Toledo et al., Dev. Cell, 2014). One key implication from our work is that oncogenic transformation leads to added requirement for BuGZ function. Further, cancer requirement for BuGZ may be more widespread than BubR1 based on our assessment in brain tumor isolates. For example, isolates that are resistant to BubR1-GLEBs domain inhibition are still sensitive to loss of BuGZ. As with BubR1, we are working with Jim Olson's group to identify small molecules or peptides that will interfere with BuGZ's GLEBs domain activity

Inhibition of PHF5A causes human GBM tumor regression and survival in immune compromised mice

PHF5A. PHF5A is a highly conserved plant homeodomain (PHD)-zinc finger domain protein that facilitates interactions between the U2 small nuclear ribonucleoprotein (snRNP) complex and DNA/RNA helicases. We found that in GSCs, but not in untransformed controls, PHF5A facilitates recognition of exons with unusual C-rich 3' splice sites (ss) in thousands of essential genes. PHF5A knockdown in GSCs, but not in untransformed NSCs, astrocytes, or fibroblasts, inhibited splicing of these genes, leading to cell cycle arrest and loss of viability. Further, induction of knockdown of PHF5A in patient-derived xenograft brain tumors in mice led to highly significant survival benefits (Hubert et al., Genes and Dev., 2013). We are currently working with Jim Olson's and Marc Ferrer's groups (Fred Hutch and NIH/NCATs) to identify small molecules that trigger splicing defects similar to PHF5A inhibition in GBM cells.

CRISPR-Cas9 genome-wide lethality screens

PKMYT1 kinase activity. Human PKMYT1/Myt1 encodes a dual specificity (threonine and tyrosine) protein kinase homologous to WEE1 that localizes to the endoplasmic reticulum-Golgi complex and, at least in vitro, can inhibit CyclinB/CDK1 activity, by phosphorylating CDK1's ATP binding domain at Thr14. PKMYT1 arose as a key GSC-lethal hit in recent genome-wide CRISPR-Cas9 screens. Mechanistic studies showed that in non-transformed cells PKMYT1 acts redundantly with WEE1 to facilitate proper mitotic entry in primary neural progenitors via phosphorylating CDK1-Thr14 and -Tyr15, which inhibits CyclinB/CDK1 activity. However, in GSCs and NPCs with activated EGFR and AKT1 the redundancy is broken causing PKMYT1 to become essential (Toledo et al., Cell Reports, 2015)

Near-term directions. We are currently comprehensively retesting all possible CRISPR-Cas9 and RNAi hits from each of our focus set and genome-wide screens using custom made lentiviral libraries. This will identify top scoring cancer-lethal genes. These targets and each of our published candidate therapeutic targets are in the process of being evaluated by our collaborators Drs. Jim Olson (Clinical Research Division) and Eric Holland (Human Biology Division) for clinical translation. Part of this evaluation process involves generating mouse models of target inhibition (e.g., inducible shRNA transgenics or Floxed allele) to examine "therapeutic window" in the context of mouse models of cancer (e.g., glioma) and also to examine specific on-target liabilities (i.e., are any normal tissues affected by inhibition?). Another arm of the effort includes generation of gene activity reporter assays for high throughput screens for molecular inhibitors of our cancer lethal targets (led by Dr. Drew Mhyre in the Olson Lab).