SUMMARY To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation.

We have designed MI-219 as a potent, highly selective and orally active small-molecule inhibitor of the MDM2-p53 interaction. MI-219 binds to human MDM2 with a Ki value of 5 nM and is 10,000-fold selective for MDM2 over MDMX. It disrupts the MDM2-p53 interaction and activates the p53 pathway in cells with wild-type p53, which leads to induction of cell cycle arrest in all cells and selective apoptosis in tumor cells. MI-219 stimulates rapid but transient p53 activation in established tumor xenograft tissues, resulting in inhibition of cell proliferation, induction of apoptosis, and complete tumor growth inhibition. MI-219 activates p53 in normal tissues with minimal p53 accumulation and is not toxic to animals. MI-219 warrants clinical investigation as a new agent for cancer treatment.cancer therapy ͉ MDM2-p53 protein-protein interaction ͉ selective toxicity to tumors ͉ small-molecule inhibitor T he tumor suppressor p53 plays a central role in the regulation of cell cycle, apoptosis, DNA repair, and senescence (1-4). Because of the prominent role played by p53 in suppressing oncogenesis (5), it is not surprising that p53 function is impaired in all human cancers. Several distinct approaches have been pursued to restore p53 function as a new cancer therapeutic strategy (6-9). Three recent studies, using unique genetic mouse models, have demonstrated that the restoration of p53 leads universally to a rapid and robust regression of established sarcomas, lymphomas, and liver tumors (10)(11)(12)(13)(14). These studies provide strong evidence that established tumors remain persistently vulnerable to p53 tumorsuppressor function and that restoration of p53 function is therefore a powerful cancer therapeutic strategy (13).In Ϸ50% of human cancers, the gene encoding p53 is either deleted or mutated, rendering the p53 protein inactive (5, 15). In the remaining cancers, p53 retains its wild-type status but its function is effectively inhibited by its primary cellular inhibitor, the human MDM2 oncoprotein (mouse double minute 2, also termed HDM2 in humans) (5,16,17). One attractive pharmacological approach to p53 reactivation is to use a small molecule to block the MDM2-p53 interaction (6)(7)(8)18). The discovery of the Nutlins provided the important proof of the concept for this approach (7). Nutlins were shown to bind to MDM2, block the MDM2-p53 interaction, and activate wild-type p53 (7,(19)(20)(21). Nutlin-3a exhibits strong anti-tumor activity in multiple xenograft mouse models of human cancer (7,19). The discovery of the Nutlins has fueled enthusiasm for the development of small-molecule MDM2 inhibitors as a new class of anticancer therapy (6,8,22,23).One critical question in the development of MDM2 inhibitors for cancer treatment is their potential toxicity to normal tissues. This concern was heightened by a recent genetic study, which showed that p53 activation in the absence of the MDM2 gene causes severe toxicity to radiosensitive normal adult mouse tissues, leading to rapid animal death (24). Previous studies on ...

Signal transducer and activator of transcription 3 (STAT3) is an attractive cancer therapeutic target. Here we report the discovery of SD-36, a small-molecule degrader of STAT3. SD-36 potently induces the degradation of STAT3 protein in vitro and in vivo and demonstrates high selectivity over other STAT members. Induced degradation of STAT3 results in a strong suppression of its transcription network in leukemia and lymphoma cells. SD-36 inhibits the growth of a subset of acute myeloid leukemia and anaplastic large-cell lymphoma cell lines by inducing cell-cycle arrest and/or apoptosis. SD-36 achieves complete and long-lasting tumor regression in multiple xenograft mouse models at well-tolerated dose schedules. Degradation of STAT3 protein, therefore, is a promising cancer therapeutic strategy.

Small-molecule Smac mimetics are being developed as a novel class of anticancer drugs. Recent studies have shown that Smac mimetics target cellular inhibitor of apoptosis protein (cIAP)-1/2 for degradation and induce tumor necrosis factor-A (TNFA)-dependent apoptosis in tumor cells. In this study, we have investigated the mechanism of action and therapeutic potential of two different types of novel Smac mimetics, monovalent SM-122 and bivalent SM-164. Our data showed that removal of cIAP-1/2 by Smac mimetics or small interfering RNA is not sufficient for robust TNFA-dependent apoptosis induction, and X-linked inhibitor of apoptosis protein (XIAP) plays a critical role in inhibiting apoptosis induction. Although SM-164 is modestly more effective than SM-122 in induction of cIAP-1/2 degradation, SM-164 is 1,000 times more potent than SM-122 as an inducer of apoptosis in tumor cells, which is attributed to its much higher potency in binding to and antagonizing XIAP. SM-164 induces rapid cIAP-1 degradation and strong apoptosis in the MDA-MB-231 xenograft tumor tissues and achieves tumor regression, but has no toxicity in normal mouse tissues. Our study provides further insights into the mechanism of action for Smac mimetics and regulation of apoptosis by inhibitor of apoptosis proteins. Furthermore, our data provide evidence that SM-164 is a promising new anticancer drug for further evaluation and development. [Cancer Res 2008;68(22):9384-93]

The bromodomain and extra-terminal (BET) family proteins, consisting of BRD2, BRD3, BRD4, and testis-specific BRDT members, are epigenetic “readers” and play a key role in the regulation of gene transcription. BET proteins are considered to be attractive therapeutic targets for cancer and other human diseases. Recently, heterobifunctional small-molecule BET degraders have been designed based upon the proteolysis targeting chimera (PROTAC) concept to induce BET protein degradation. Herein, we present our design, synthesis, and evaluation of a new class of PROTAC BET degraders. One of the most promising compounds, 23, effectively degrades BRD4 protein at concentrations as low as 30 pM in the RS4;11 leukemia cell line, achieves an IC50 value of 51 pM in inhibition of RS4;11 cell growth and induces rapid tumor regression in vivo against RS4;11 xenograft tumors. These data establish that compound 23 (BETd-260/ZBC260) is a highly potent and efficacious BET degrader.

Blocking the MDM2-p53 protein-protein interaction has long been considered to offer a broad cancer therapeutic strategy, despite the potential risks of selecting tumors harboring p53 mutations that escape MDM2 control. In this study, we report a novel small molecule inhibitor of the MDM2-p53 interaction, SAR405838 (MI-77301) that has been advanced into Phase I clinical trials. SAR405838 binds to MDM2 with Ki = 0.88 nM and has high specificity over other proteins. A co-crystal structure of the SAR405838:MDM2 complex shows that in addition to mimicking three key p53 amino acid residues, the inhibitor captures additional interactions not observed in the p53-MDM2 complex and induces refolding of the short, unstructured MDM2 N-terminal region to achieve its high affinity. SAR405838 effectively activates wild-type p53 in vitro and in xenograft tumor tissue of leukemia and solid tumors, leading to p53-dependent cell cycle arrest and/or apoptosis. At well-tolerated dose schedules, SAR405838 achieves either durable tumor regression or complete tumor growth inhibition in mouse xenograft models of SJSA-1 osteosarcoma, RS4;11 acute leukemia, LNCaP prostate cancer and HCT-116 colon cancer. Remarkably, a single oral dose of SAR405838 is sufficient to achieve complete tumor regression in the SJSA-1 model. Mechanistically, robust transcriptional up-regulation of PUMA induced by SAR405838 results in strong apoptosis in tumor tissue, leading to complete tumor regression. Our findings provide a preclinical basis upon which to evaluate SAR405838 as a therapeutic agent in patients whose tumors retain wild-type p53.

We report the discovery and characterization of SM-406 (compound 2), a potent and orally bioavailable Smac mimetic and an antagonist of the inhibitor of apoptosis proteins (IAPs). This compound binds to XIAP, cIAP1 and cIAP2 proteins with Ki values of 66.4 nM, 1.9 nM and 5.1 nM, respectively. Compound 2 effectively antagonizes XIAP BIR3 protein in a cell-free functional assay, induces rapid degradation of cellular cIAP1 protein and inhibits cancer cell growth in various human cancer cell lines. It has good oral bioavailability in mice, rats, non-human primates and dogs, is highly effective in induction of apoptosis in xenograft tumors and is capable of complete inhibition of tumor growth. Compound 2 is currently in Phase I clinical trials for the treatment of human cancer.

Proteins of the bromodomain and extra-terminal (BET) family are epigenetics "readers" and promising therapeutic targets for cancer and other human diseases. We describe herein a structure-guided design of [1,4]oxazepines as a new class of BET inhibitors and our subsequent design, synthesis, and evaluation of proteolysis-targeting chimeric (PROTAC) small-molecule BET degraders. Our efforts have led to the discovery of extremely potent BET degraders, exemplified by QCA570, which effectively induces degradation of BET proteins and inhibits cell growth in human acute leukemia cell lines even at low picomolar concentrations. QCA570 achieves complete and durable tumor regression in leukemia xenograft models in mice at well-tolerated dose-schedules. QCA570 is the most potent and efficacious BET degrader reported to date.