Elisabeth Lasater

Dual kinase-bromodomain inhibitors for rationally designed polypharmacology.

Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multi-target profile has, however, necessitated the application of combination therapies, which can pose significant clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as novel targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase/bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348, clinical PLK1 and JAK2/FLT3 kinase inhibitors, respectively, is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a novel strategy for rational single agent polypharmacological targeting. Furthermore, structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase/bromodomain inhibitors.

Crenolanib is a selective type I pan-FLT3 inhibitor

Significance Rapid evolution of drug resistance associated with secondary kinase domain (KD) mutations is the best characterized mechanism of acquired resistance to effective tyrosine kinase inhibitor (TKI) therapy. Medicinal chemistry efforts have largely been devoted toward synthesizing type II TKIs that, by targeting an inactive kinase conformation, are believed to afford greater selectivity than type I TKIs that bind an active kinase conformation. The only previously described TKI with the ability to successfully suppress all resistance-conferring KD mutants (i.e. “pan-kinase” inhibitor) is the type II multikinase TKI ponatinib. Here, we demonstrate that a type I TKI can be potent, selective, and invulnerable to resistance-conferring KD mutation as a mechanism of resistance. Efforts to develop potent, selective type I pan-kinase inhibitors are warranted. Tyrosine kinase inhibitors (TKIs) represent transformative therapies for several malignancies. Two critical features necessary for maximizing TKI tolerability and response duration are kinase selectivity and invulnerability to resistance-conferring kinase domain (KD) mutations in the intended target. No prior TKI has demonstrated both of these properties. Aiming to maximize selectivity, medicinal chemists have largely sought to create TKIs that bind to an inactive (type II) kinase conformation. Here we demonstrate that the investigational type I TKI crenolanib is a potent inhibitor of Fms tyrosine kinase-3 (FLT3) internal tandem duplication, a validated therapeutic target in human acute myeloid leukemia (AML), as well as all secondary KD mutants previously shown to confer resistance to the first highly active FLT3 TKI quizartinib. Moreover, crenolanib is highly selective for FLT3 relative to the closely related protein tyrosine kinase KIT, demonstrating that simultaneous FLT3/KIT inhibition, a prominent feature of other clinically active FLT3 TKIs, is not required for AML cell cytotoxicity in vitro and may contribute to undesirable toxicity in patients. A saturation mutagenesis screen of FLT3–internal tandem duplication failed to recover any resistant colonies in the presence of a crenolanib concentration well below what has been safely achieved in humans, suggesting that crenolanib has the potential to suppress KD mutation-mediated clinical resistance. Crenolanib represents the first TKI to exhibit both kinase selectivity and invulnerability to resistance-conferring KD mutations, which is unexpected of a type I inhibitor. Crenolanib has significant promise for achieving deep and durable responses in FLT3–mutant AML, and may have a profound impact upon future medicinal chemistry efforts in oncology.

MEK-Dependent Negative Feedback Underlies BCR–ABL-Mediated Oncogene Addiction

UNLABELLED The clinical experience with BCR-ABL tyrosine kinase inhibitors (TKI) for the treatment of chronic myelogenous leukemia (CML) provides compelling evidence for oncogene addiction. Yet, the molecular basis of oncogene addiction remains elusive. Through unbiased quantitative phosphoproteomic analyses of CML cells transiently exposed to BCR-ABL TKI, we identified persistent downregulation of growth factor receptor (GF-R) signaling pathways. We then established and validated a tissue-relevant isogenic model of BCR-ABL-mediated addiction, and found evidence for myeloid GF-R signaling pathway rewiring that profoundly and persistently dampens physiologic pathway activation. We demonstrate that eventual restoration of ligand-mediated GF-R pathway activation is insufficient to fully rescue cells from a competing apoptotic fate. In contrast to previous work with BRAF(V600E) in melanoma cells, feedback inhibition following BCR-ABL TKI treatment is markedly prolonged, extending beyond the time required to initiate apoptosis. Mechanistically, BCR-ABL-mediated oncogene addiction is facilitated by persistent high levels of MAP-ERK kinase (MEK)-dependent negative feedback. SIGNIFICANCE We found that BCR–ABL can confer addiction in vitro by rewiring myeloid GF-R signaling through establishment of MEK-dependent negative feedback. Our findings predict that deeper, more durable responses to targeted agents across a range of malignancies may be facilitated by maintaining negative feedback concurrently with oncoprotein inhibition.

Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy

Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a ‘synthetic lethal toxicity’ arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity. DOI: http://dx.doi.org/10.7554/eLife.03445.001

The MERTK/FLT3 inhibitor MRX-2843 overcomes resistance-conferring FLT3 mutations in acute myeloid leukemia

FMS-like tyrosine kinase 3-targeted (FLT3-targeted) therapies have shown initial promise for the treatment of acute myeloid leukemia (AML) expressing FLT3-activating mutations; however, resistance emerges rapidly. Furthermore, limited options exist for the treatment of FLT3-independent AML, demonstrating the need for novel therapies that reduce toxicity and improve survival. MERTK receptor tyrosine kinase is overexpressed in 80% to 90% of AMLs and contributes to leukemogenesis. Here, we describe MRX-2843, a type 1 small-molecule tyrosine kinase inhibitor that abrogates activation of both MERTK and FLT3 and their downstream effectors. MRX-2843 treatment induces apoptosis and inhibits colony formation in AML cell lines and primary patient samples expressing MERTK and/or FLT3-ITD, with a wide therapeutic window compared with that of normal human cord blood cells. In murine orthotopic xenograft models, once-daily oral therapy prolonged survival 2- to 3-fold over that of vehicle-treated controls. Additionally, MRX-2843 retained activity against quizartinib-resistant FLT3-ITD-mutant proteins with clinically relevant alterations at the D835 or F691 loci and prolonged survival in xenograft models of quizartinib-resistant AML. Together, these observations validate MRX-2843 as a translational agent and support its clinical development for the treatment of AML.

Abstract 1893: Activating NRAS mutations are associated with FLT3-independent resistance to the clinically active FLT3 inhibitor AC220 in vitro

Background: Clinical outcomes associated with acute myeloid leukemia (AML) have not improved substantially in decades. Activating FMS-like tyrosine kinase-3 (FLT3) mutations are detected in ∼30% of AML cases with internal tandem duplication (ITD) mutations conferring worse prognosis. Recently, the potent FLT3 inhibitor AC220 has achieved composite complete remission in 45% of patients with relapsed/refractory FLT3-mutant AML (Cortes et al, EHA 2011). Translational studies from our laboratory implicate reactivation of FLT3-ITD through kinase domain mutations in the majority AC220 relapse cases (Smith et al, ASH 2011). Given that less than half of treated patients initially achieve deep remissions on AC220, we hypothesize that off-target (non-FLT3 mediated) resistance is responsible for primary resistance to AC220, and may mediate loss of response in a subset of patients. Results: To model and identify putative mechanisms of off-target resistance to AC220 in vitro, we cultured the FLT3-ITD+ patient derived cell lines MV4;11 and Molm14 in escalating concentrations of AC220. To date, we have isolated 8 Molm14 subclones that are resistant to at least 20nM AC220, which is >60-fold the IC50 of the parental line (0.3nM), while efforts to generate resistant MV4;11 cells have repeatedly failed. Analysis of FLT3 signaling revealed persistent FLT3 phosphorylation in 5 clones in the presence of 10nM AC220. Sequencing of the FLT3 kinase domain identified acquired secondary activation loop mutations suggesting on-target acquired resistance. The remaining 3 clones demonstrated off-target resistance as evidenced by the absence of a FLT3 mutation and loss of FLT3 phosphorylation with 10nM AC220 treatment. Despite FLT3 inhibition, the clones failed to undergo apoptosis and maintained ERK phosphorylation when treated with AC220. Sequencing of K- and NRAS revealed an NRAS G12C mutation in 2 clones and an NRAS Q61K mutation in the third clone, both of which are known activating RAS mutations. The AC220 IC50 values for the G12C mutant clones were determined to be 134nM and 329nM AC220 and the Q61K mutant was one of the most resistant of all clones with an IC50 of >1000nM AC220. Further, all 3 NRAS mutant clones showed increased sensitivity to the MEK inhibitor PD0325901 compared to the parental cells and on-target resistant clones. Conclusions: Generation of 8 independent AC220-resistant AML cell lines revealed evidence for on- or off-target resistance mechanisms. We have identified oncogenic RAS as a putative mechanism of acquired AC220 off-target resistance in vitro, suggesting that pathologic activation of the RAS/ERK pathway may result in failure of clinically active FLT3 inhibitor therapy in AML patients. Assessment for activating RAS mutations in patient samples with primary and acquired resistance to AC220 and other clinically effective FLT3 inhibitors is currently ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1893. doi:1538-7445.AM2012-1893

Abstract 2923: Phosphoproteomic assessment of CML cells following transient potent inhibition with dasatinib is associated with durable alteration of the STAT5 and RAF/MEK/ERK pathways

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The critical reliance of CML cells upon BCR-ABL kinase activity for survival supports the concept of “oncogene addiction”, but the molecular mechanisms underlying this phenomenon are poorly understood. Consistent with clinical responses in CML patients to once daily administration of dasatinib, we have previously demonstrated that transient, but potent tyrosine kinase inhibition of BCR-ABL is sufficient to induce cytotoxicity of the CML cell line K562 in vitro. These cells display an irreversible commitment to apoptosis despite reactivation of BCR-ABL kinase activity as assessed by rephosphorylation of the substrate CRKL. We hypothesized that durable alterations in signaling occur in this in vitro model of transient kinase inhibition and that a subset of these changes are critical to eliciting cytotoxicity. To obtain an unbiased, global view of signaling before and after a high-dose pulse (HDP) of dasatinib, we optimized phosphoproteomic technology to interrogate changes in the phosphotyrosine proteome of K562 cells. Through implementation of SILAC to assess quantitative changes in tyrosine phosphorylation and a high pH C18 fractionation prior to MS/MS analysis, we compared lysates from the end-of-exposure, 3 hours, and 6 hours post-HDP, and identified 204 unique phosphosites originating from 160 different proteins. Notably, following transient exposure to dasatinib, durable alterations in phosphorylation occurred in two of the three major signaling pathways downstream of BCR-ABL. Specifically, we observed a durable loss of tyrosine phosphorylation in the SH2-domain of STAT5, as well as a loss of phosphorylation within the activation loop of ERK1/2 without alterations in the PI3K/AKT pathway. We have confirmed these findings with phospho-specific antibodies. Of particular interest is the mechanism whereby the STAT5 and RAS/MAPK signaling pathways are durably altered. Interestingly, despite durable alterations in the phosphorylation status of MEK, ERK and BIM, the overall level of activated RAS is unaltered with dasatinib treatment. We therefore hypothesize that the RAF kinases are durably altered and are presently investigating the molecular mechanism responsible. Lastly, despite durable loss of STAT5 phosphorylation we observed no activation-loop phosphorylation of JAK2 before or after a HDP, suggestive of a JAK2-independent mechanism of STAT5 activation in K562 cells. Our findings suggest that the molecular basis of oncogene addition in CML involves irreversible hijacking of the STAT5 and RAF/MEK/ERK pathways by BCR-ABL as evidenced by durable loss of signaling through these pathways following potent transient BCR-ABL inhibition. Current work is evaluating the molecular mechanisms that underlie these durable signaling alterations. MS analysis was provided by the MS Resource at UCSF (NIH NCRR P41RR001614). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2923. doi:10.1158/1538-7445.AM2011-2923