The Irreversible Covalent Fibroblast Growth Factor Receptor Inhibitor PRN1371 Exhibits Sustained Inhibition of FGFR after Drug Clearance.

An increasing number of cancers are known to harbor mutations, translocations, or amplifications in the fibroblast growth factor receptor (FGFR) family of kinases. The FGFR inhibitors evaluated in clinical trials to date have shown promise at treating these cancers. Here, we describe PRN1371, an irreversible covalent inhibitor of FGFR1-4 targeting a cysteine within the kinase active site. PRN1371 demonstrated strong FGFR potency and excellent kinome-wide selectivity in a number of biochemical and cellular assays, including in various cancer cell lines exhibiting FGFR alterations. Furthermore, PRN1371 maintained FGFR inhibition in vivo, not only when circulating drug levels were high but also after the drug had been cleared from circulation, indicating the possibility of sustained FGFR inhibition in the clinic without the need for continuous drug exposure. Durable tumor regression was also obtained in multiple tumor xenografts and patient-derived tumor xenograft models and was sustained even using an intermittent dosing strategy that provided drug holidays. PRN1371 is currently under clinical investigation for treatment of patients with solid tumors. Mol Cancer Ther; 16(12); 2668-76. ©2017 AACR.

Discovery of the Irreversible Covalent FGFR Inhibitor 8-(3-(4-Acryloylpiperazin-1-yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (PRN1371) for the Treatment of Solid Tumors.

Aberrant signaling of the FGF/FGFR pathway occurs frequently in cancers and is an oncogenic driver in many solid tumors. Clinical validation of FGFR as a therapeutic target has been demonstrated in bladder, liver, lung, breast, and gastric cancers. Our goal was to develop an irreversible covalent inhibitor of FGFR1-4 for use in oncology indications. An irreversible covalent binding mechanism imparts many desirable pharmacological benefits including high potency, selectivity, and prolonged target inhibition. Herein we report the structure-based design, medicinal chemistry optimization, and unique ADME assays of our irreversible covalent drug discovery program which culminated in the discovery of compound 34 (PRN1371), a highly selective and potent FGFR1-4 inhibitor.

Cooperative Dynamics of AR and ER Activity in Breast Cancer.

Androgen receptor (AR) is expressed in 90% of estrogen receptor alpha-positive (ER+) breast tumors, but its role in tumor growth and progression remains controversial. Use of two anti-androgens that inhibit AR nuclear localization, enzalutamide and MJC13, revealed that AR is required for maximum ER genomic binding. Here, a novel global examination of AR chromatin binding found that estradiol induced AR binding at unique sites compared with dihydrotestosterone (DHT). Estradiol-induced AR-binding sites were enriched for estrogen response elements and had significant overlap with ER-binding sites. Furthermore, AR inhibition reduced baseline and estradiol-mediated proliferation in multiple ER+/AR+ breast cancer cell lines, and synergized with tamoxifen and fulvestrant. In vivo, enzalutamide significantly reduced viability of tamoxifen-resistant MCF7 xenograft tumors and an ER+/AR+ patient-derived model. Enzalutamide also reduced metastatic burden following cardiac injection. Finally, in a comparison of ER+/AR+ primary tumors versus patient-matched local recurrences or distant metastases, AR expression was often maintained even when ER was reduced or absent. These data provide preclinical evidence that anti-androgens that inhibit AR nuclear localization affect both AR and ER, and are effective in combination with current breast cancer therapies. In addition, single-agent efficacy may be possible in tumors resistant to traditional endocrine therapy, as clinical specimens of recurrent disease demonstrate AR expression in tumors with absent or refractory ER. IMPLICATIONS: This study suggests that AR plays a previously unrecognized role in supporting E2-mediated ER activity in ER+/AR+ breast cancer cells, and that enzalutamide may be an effective therapeutic in ER+/AR+ breast cancers. Mol Cancer Res; 14(11); 1054-67. ©2016 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.

Oncogenic RAS pathway activation promotes resistance to anti-VEGF therapy through G-CSF-induced neutrophil recruitment.

Granulocyte-colony stimulating factor (G-CSF) promotes mobilization of CD11b(+)Gr1(+) myeloid cells and has been implicated in resistance to anti-VEGF therapy in mouse models. High G-CSF production has been associated with a poor prognosis in cancer patients. Here we show that activation of the RAS/MEK/ERK pathway regulates G-CSF expression through the Ets transcription factor. Several growth factors induced G-CSF expression by a MEK-dependent mechanism. Inhibition of G-CSF release with a MEK inhibitor markedly reduced G-CSF production in vitro and synergized with anti-VEGF antibodies to reduce CD11b(+)Ly6G(+) neutrophil mobilization and tumor growth and led to increased survival in animal models of cancer, including a genetically engineered mouse model of pancreatic adenocarcinoma. Analysis of biopsies from pancreatic cancer patients revealed increased phospho-MEK, G-CSF, and Ets expression and enhanced neutrophil recruitment compared with normal pancreata. These results provide insights into G-CSF regulation and on the mechanism of action of MEK inhibitors and point to unique anticancer strategies.

The RasGAP proteins Ira2 and neurofibromin are negatively regulated by Gpb1 in yeast and ETEA in humans.

The neurofibromatosis type 1 (NF1) gene encodes the GTPase-activating protein (GAP) neurofibromin, which negatively regulates Ras activity. The yeast Saccharomyces cerevisiae has two neurofibromin homologs, Ira1 and Ira2. To understand how these proteins are regulated, we utilized an unbiased proteomics approach to identify Ira2 and neurofibromin binding partners. We demonstrate that the Gpb1/Krh2 protein binds and negatively regulates Ira2 by promoting its ubiquitin-dependent proteolysis. We extended our findings to show that in mammalian cells, the ETEA/UBXD8 protein directly interacts with and negatively regulates neurofibromin. ETEA contains both UBA and UBX domains. Overexpression of ETEA downregulates neurofibromin in human cells. Purified ETEA, but not a mutant of ETEA that lacks the UBX domain, ubiquitinates the neurofibromin GAP-related domain in vitro. Silencing of ETEA expression increases neurofibromin levels and downregulates Ras activity. These findings provide evidence for conserved ubiquitination pathways regulating the RasGAP proteins Ira2 (in yeast) and neurofibromin (in humans).

Ras signaling and therapies.

More than 25 years have passed since activating mutations in Ras genes were identified in DNA from human tumors. In this time, it has been established beyond doubt that these mutations play a direct role in causing cancer, and do so in collaboration with a number of other oncogenes and tumor suppressors. Oncogenic mutant Ras proteins are resistant to downregulation by GAP-mediated hydrolysis of bound GTP, and therefore signal persistently. Efforts to develop therapies that block Ras oncoprotein function directly have failed. The high affinity of Ras proteins for GTP has discouraged attempts to identify GTP-analogs. Ras processing enzymes have been targeted, but unfortunately, K-Ras, the Ras protein that plays the major role in human cancer, has proven refractory to these approaches. Further progress has been made with drugs that block downstream signaling: the approved drug Sorafenib inhibits Raf kinase, and its clinical benefits in liver cancer are greatest in patients in which the mitogen activated protein kinase (MAPK) signaling pathway is hyperactive. Other Raf kinase inhibitors, as well as drugs that block mitogen-activated protein kinase / extracellular signal-regulated kinase kinase (MEK) and various steps in the PI 3' kinase pathway, are under development. Here we will discuss the complexities of Ras signaling and their effects on targeting the Ras pathway in the future.

Forced retinoic acid receptor alpha homodimers prime mice for APL-like leukemia.

RARA becomes an acute promyelocytic leukemia (APL) oncogene by fusion with any of five translocation partners. Unlike RARalpha, the fusion proteins homodimerize, which may be central to oncogenic activation. This model was tested by replacing PML with dimerization domains from p50NFkappaB (p50-RARalpha) or the rapamycin-sensitive dimerizing peptide of FKBP12 (F3-RARalpha). The X-RARalpha fusions recapitulated in vitro activities of PML-RARalpha. For F3-RARalpha, these properties were rapamycin sensitive. Although in vivo the artificial fusions alone are poor initiators of leukemia, p50-RARalpha readily cooperates with an activated mutant CDw131 to induce APL-like disease. These results demonstrate that the dimerization interface of RARalpha fusion partners is a critical element in APL pathogenesis while pointing to other features of PML for enhancing penetrance and progression.

Cooperation of cytokine signaling with chimeric transcription factors in leukemogenesis: PML-retinoic acid receptor alpha blocks growth factor-mediated differentiation.

We utilized a mouse model of acute promyelocytic leukemia (APL) to investigate how aberrant activation of cytokine signaling pathways interacts with chimeric transcription factors to generate acute myeloid leukemia. Expression in mice of the APL-associated fusion, PML-RARA, initially has only modest effects on myelopoiesis. Whereas treatment of control animals with interleukin-3 (IL-3) resulted in expanded myelopoiesis without a block in differentiation, PML-RARA abrogated differentiation that normally characterizes the response to IL-3. Retroviral transduction of bone marrow with an IL-3-expressing retrovirus revealed that IL-3 and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) combined to generate a lethal leukemia-like syndrome in <21 days. We also observed that a constitutively activated mutant IL-3 receptor, beta(c)V449E, cooperated with PML-RARalpha in leukemogenesis, whereas a different activated mutant, beta(c)I374N, did not. Analysis of additional mutations introduced into beta(c)V449E showed that, although tyrosine phosphorylation of beta(c) is necessary for cooperation, the Src homology 2 domain-containing transforming protein binding site is dispensable. Our results indicate that chimeric transcription factors can block the differentiative effects of growth factors. This combination can be potently leukemogenic, but the particular manner in which these types of mutations interact determines the ability of such combinations to generate acute myeloid leukemia.

Recurring chromosomal abnormalities in leukemia in PML-RARA transgenic mice identify cooperating events and genetic pathways to acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by the PML-RARA fusion gene. To identify genetic changes that cooperate with PML-RARA, we performed spectral karyotyping analysis of myeloid leukemias from transgenic PML-RARA mice and from mice coexpressing PML-RARA and BCL2, IL3, activated IL3R, or activated FLT3. A cooperating mutation that enhanced survival (BCL2) was not sufficient to complete transformation and was associated with multiple numeric abnormalities, whereas cooperating mutations that deregulated growth and enhanced survival were associated with normal karyotypes (IL3) or simple karyotypic changes (IL3R, FLT3). Recurring abnormalities included trisomy 15 (49%), trisomy 8 (46%), and -X/-Y (54%). The most common secondary abnormality in human APL is +8 or partial trisomy of 8q24, syntenic to mouse 15. These murine leukemias have a defined spectrum of changes that recapitulates, in part, the cytogenetic abnormalities found in human APL. Our results demonstrate that different cooperating events may generate leukemia via different pathways.

A model of APL with FLT3 mutation is responsive to retinoic acid and a receptor tyrosine kinase inhibitor, SU11657.

The PML-RAR alpha fusion protein is central to the pathogenesis of acute promyelocytic leukemia (APL). Expression of this protein in transgenic mice initiates myeloid leukemias with features of human APL, but only after a long latency (8.5 months in MRP8 PML-RARA mice). Thus, additional changes contribute to leukemic transformation. Activating mutations of the FLT3 receptor tyrosine kinase are common in human acute myeloid leukemias and are frequent in human APL. To assess how activating mutations of FLT3 contribute to APL pathogenesis and impact therapy, we used retroviral transduction to introduce an activated allele of FLT3 into control and MRP8 PML-RARA transgenic bone marrow. Activated FLT3 cooperated with PML-RAR alpha to induce leukemias in 62 to 299 days (median latency, 105 days). In contrast to the leukemias that arose spontaneously in MRP8 PML-RARA mice, the activated FLT3/PML-RAR alpha leukemias were characterized by leukocytosis, similar to human APL with FLT3 mutations. Cytogenetic analysis revealed clonal karyotypic abnormalities, which may contribute to pathogenesis or progression. SU11657, a selective, oral, multitargeted tyrosine kinase inhibitor that targets FLT3, cooperated with all-trans retinoic acid to rapidly cause regression of leukemia. Our results suggest that the acquisition of FLT3 mutations by cells with a pre-existing t(15;17) is a frequent pathway to the development of APL. Our findings also indicate that APL patients with FLT3 mutations may benefit from combination therapy with all-trans retinoic acid plus an FLT3 inhibitor.