Discovery of Reversible Covalent Bruton's Tyrosine Kinase Inhibitors PRN473 and PRN1008 (Rilzabrutinib).

Bruton's tyrosine kinase (BTK), a Tec family tyrosine kinase, is critical in immune pathways as an essential intracellular signaling element, participating in both adaptive and immune responses. Currently approved BTK inhibitors are irreversible covalent inhibitors and limited to oncology indications. Herein, we describe the design of covalent reversible BTK inhibitors and the discoveries of PRN473 (11) and rilzabrutinib (PRN1008, 12). These compounds have exhibited potent and durable inhibition of BTK, in vivo efficacy in rodent arthritis models, and clinical efficacy in canine pemphigus foliaceus. Compound 11 has completed phase 1 trials as a topical agent, and 12 is in phase 3 trials for pemphigus vulgaris and immune thrombocytopenia.

Single-cell transcriptomics of the naked mole-rat reveals unexpected features of mammalian immunity.

The immune system comprises a complex network of specialized cells that protects against infection, eliminates cancerous cells, and regulates tissue repair, thus serving a critical role in homeostasis, health span, and life span. The subterranean-dwelling naked mole-rat (NM-R; Heterocephalus glaber) exhibits prolonged life span relative to its body size, is unusually cancer resistant, and manifests few physiological or molecular changes with advancing age. We therefore hypothesized that the immune system of NM-Rs evolved unique features that confer enhanced cancer immunosurveillance and prevent the age-associated decline in homeostasis. Using single-cell RNA-sequencing (scRNA-seq) we mapped the immune system of the NM-R and compared it to that of the short-lived, cancer-prone mouse. In contrast to the mouse, we find that the NM-R immune system is characterized by a high myeloid-to-lymphoid cell ratio that includes a novel, lipopolysaccharide (LPS)-responsive, granulocyte cell subset. Surprisingly, we also find that NM-Rs lack canonical natural killer (NK) cells. Our comparative genomics analyses support this finding, showing that the NM-R genome lacks an expanded gene family that controls NK cell function in several other species. Furthermore, we reconstructed the evolutionary history that likely led to this genomic state. The NM-R thus challenges our current understanding of mammalian immunity, favoring an atypical, myeloid-biased mode of innate immunosurveillance, which may contribute to its remarkable health span.

Constitutive NOTCH3 Signaling Promotes the Growth of Basal Breast Cancers.

Notch ligands signal through one of four receptors on neighboring cells to mediate cell-cell communication and control cell fate, proliferation, and survival. Although aberrant Notch activation has been implicated in numerous malignancies, including breast cancer, the importance of individual receptors in distinct breast cancer subtypes and the mechanisms of receptor activation remain unclear. Using a novel antibody to detect active NOTCH3, we report here that NOTCH3 signals constitutively in a panel of basal breast cancer cell lines and in more than one third of basal tumors. Selective inhibition of individual ligands revealed that this signal does not require canonical ligand induction. A NOTCH3 antagonist antibody inhibited growth of basal lines, whereas a NOTCH3 agonist antibody enhanced the transformed phenotype in vitro and in tumor xenografts. Transcriptomic analyses generated a Notch gene signature that included Notch pathway components, the oncogene c-Myc, and the mammary stem cell regulator Id4 This signature drove clustering of breast cancer cell lines and tumors into the common subtypes and correlated with the basal classification. Our results highlight an unexpected ligand-independent induction mechanism and suggest that constitutive NOTCH3 signaling can drive an oncogenic program in a subset of basal breast cancers. Cancer Res; 77(6); 1439-52. ©2017 AACR.

Prolonged and tunable residence time using reversible covalent kinase inhibitors.

Drugs with prolonged on-target residence times often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here we made progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Using an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrated biochemical residence times spanning from minutes to 7 d. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK for more than 18 h after clearance from the circulation. The inverted cyanoacrylamide strategy was further used to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating the generalizability of the approach. Targeting of noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates 'residence time by design', the ability to modulate and improve the duration of target engagement in vivo.

Mapping in vivo tumor oxygenation within viable tumor by 19F-MRI and multispectral analysis.

Quantifying oxygenation in viable tumor remains a major obstacle toward a better understanding of the tumor micro-environment and improving treatment strategies. Current techniques are often complicated by tumor heterogeneity. Herein, a novel in vivo approach that combines (19)F magnetic resonance imaging ((19)F-MRI) R 1 mapping with diffusion-based multispectral (MS) analysis is introduced. This approach restricts the partial pressure of oxygen (pO2) measurements to viable tumor, the tissue of therapeutic interest. The technique exhibited sufficient sensitivity to detect a breathing gas challenge in a xenograft tumor model, and the hypoxic region measured by MS (19)F-MRI was strongly correlated with histologic estimates of hypoxia. This approach was then applied to address the effects of antivascular agents on tumor oxygenation, which is a research question that is still under debate. The technique was used to monitor longitudinal pO2 changes in response to an antibody to vascular endothelial growth factor (B20.4.1.1) and a selective dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor (GDC-0980). GDC-0980 reduced viable tumor pO2 during a 3-day treatment period, and a significant reduction was also produced by B20.4.1.1. Overall, this method provides an unprecedented view of viable tumor pO2 and contributes to a greater understanding of the effects of antivascular therapies on the tumor's microenvironment.

Differential drug class-specific metastatic effects following treatment with a panel of angiogenesis inhibitors.

Inhibiting angiogenesis has become an important therapeutic strategy for cancer treatment but, like other current targeted therapies, benefits experienced for late-stage cancers can be curtailed by inherent refractoriness or by acquired drug resistance, requiring a need for better mechanistic understanding of such effects. Numerous preclinical studies have demonstrated that VEGF pathway inhibitors suppress primary tumour growth and metastasis. However, it has been recently reported that short-term VEGF and VEGFR inhibition can paradoxically accelerate tumour invasiveness and metastasis in certain models. Here we comprehensively compare the effects of both antibody and small molecule receptor tyrosine kinase (RTK) inhibitors targeting the VEGF-VEGFR pathway, using short-term therapy in various mouse models of metastasis. Our findings demonstrate that antibody inhibition of VEGF pathway molecules does not promote metastasis, in contrast to selected small molecule RTK inhibitors at elevated-therapeutic drug dosages. In particular, a multi-targeted RTK inhibitor, sunitinib, which most profoundly potentiated metastasis, also increased lung vascular permeability and promoted tumour cell extravasation. Mechanistically, sunitinib, but not anti-VEGF treatment, attenuated endothelial barrier function in culture and caused a global inhibition of protein tyrosine phosphorylation, including molecules important for maintaining endothelial cell-cell junctions. Together these findings indicate that, rather than a specific consequence of inhibiting the VEGF signalling pathway, pharmacological inhibitors of the VEGF pathway can have dose- and drug class-dependent side-effects on the host vasculature. These findings also advocate for the continued identification of mechanisms of resistance to anti-angiogenics and for therapy development to overcome it.

Therapeutic antibody targeting of individual Notch receptors.

The four receptors of the Notch family are widely expressed transmembrane proteins that function as key conduits through which mammalian cells communicate to regulate cell fate and growth. Ligand binding triggers a conformational change in the receptor negative regulatory region (NRR) that enables ADAM protease cleavage at a juxtamembrane site that otherwise lies buried within the quiescent NRR. Subsequent intramembrane proteolysis catalysed by the gamma-secretase complex liberates the intracellular domain (ICD) to initiate the downstream Notch transcriptional program. Aberrant signalling through each receptor has been linked to numerous diseases, particularly cancer, making the Notch pathway a compelling target for new drugs. Although gamma-secretase inhibitors (GSIs) have progressed into the clinic, GSIs fail to distinguish individual Notch receptors, inhibit other signalling pathways and cause intestinal toxicity, attributed to dual inhibition of Notch1 and 2 (ref. 11). To elucidate the discrete functions of Notch1 and Notch2 and develop clinically relevant inhibitors that reduce intestinal toxicity, we used phage display technology to generate highly specialized antibodies that specifically antagonize each receptor paralogue and yet cross-react with the human and mouse sequences, enabling the discrimination of Notch1 versus Notch2 function in human patients and rodent models. Our co-crystal structure shows that the inhibitory mechanism relies on stabilizing NRR quiescence. Selective blocking of Notch1 inhibits tumour growth in pre-clinical models through two mechanisms: inhibition of cancer cell growth and deregulation of angiogenesis. Whereas inhibition of Notch1 plus Notch2 causes severe intestinal toxicity, inhibition of either receptor alone reduces or avoids this effect, demonstrating a clear advantage over pan-Notch inhibitors. Our studies emphasize the value of paralogue-specific antagonists in dissecting the contributions of distinct Notch receptors to differentiation and disease and reveal the therapeutic promise in targeting Notch1 and Notch2 independently.

HER2-targeted therapy reduces incidence and progression of midlife mammary tumors in female murine mammary tumor virus huHER2-transgenic mice.

PURPOSE: This study examined the effectiveness of early and prolonged mu4D5 (the murine form of trastuzumab/Herceptin) treatment in transgenic mice that overexpress human HER2 (huHER2), under the murine mammary tumor virus promoter, as a model of huHER2-overexpressing breast cancer. EXPERIMENTAL DESIGN: Mice were randomly assigned to one of three treatment groups and received i.p. injections from 17 weeks of age until either 52 weeks of age or morbidity. Fourteen mice received 100 mg/kg mu4D5, 14 mice received 100 mg/kg antiherpes simplex virus glycoprotein D control antibody, and 11 mice received a diluent control. RESULTS: High levels of huHER2 expression were detectable in mammary glands of young virgin founder mice. Mammary adenocarcinomas were frequently found in female founders and progeny at an average age of 28 weeks, with some progressing to metastatic disease. The incidence of mammary tumors was significantly reduced, and tumor growth inhibition was observed in mice receiving mu4D5 compared with control mice. In addition, Harderian gland neoplasms, highly associated with overexpression of huHER2 in this transgenic line, were entirely absent in the mu4D5 treatment group, indicating down-regulation of huHER2 in vivo activity. CONCLUSIONS: Early intervention with mu4D5 was of benefit in our transgenic mice at high risk for developing huHER2-overexpressing breast cancer. This study suggests a potential benefit of early treatment with Herceptin in HER2-positive primary breast cancer.