Christine Sastri

Discovery of 5-(1H-indol-5-yl)-1,3,4-thiadiazol-2-amines as potent PIM inhibitors.

PIM kinases are a family of Ser/Thr kinases that are implicated in tumorigenesis. The discovery of a new class of PIM inhibitors, 5-(1H-indol-5-yl)-1,3,4-thiadiazol-2-amines, is discussed with optimized compounds showing excellent potency against all three PIM isoforms.

The discovery and optimization of aminooxadiazoles as potent Pim kinase inhibitors.

High levels of Pim expression have been implicated in several hematopoietic and solid tumor cancers. These findings suggest that inhibition of Pim signaling by a small molecule Pim-1,2 inhibitor could provide patients with therapeutic benefit. Herein, we describe our progress towards this goal starting from the highly Pim-selective indole-thiadiazole compound (1), which was derived from a nonselective hit identified in a high throughput screening campaign. Optimization of this compounds potency and its pharmacokinetic properties resulted in the discovery of compound 29. Cyclopropane 29 was found to exhibit excellent enzymatic potency on the Pim-1 and Pim-2 isoforms (Ki values of 0.55nM and 0.28nM, respectively), and found to inhibit the phosphorylation of BAD in the Pim-overexpressing KMS-12 cell line (IC50=150nM). This compound had moderate clearance and bioavailability in rat (CL=2.42L/kg/h; %F=24) and exhibited a dose-dependent inhibition of p-BAD in KMS-12 tumor pharmacodynamic (PD) model with an EC50 value of 6.74μM (18μg/mL) when dosed at 10, 30, 100 and 200mg/kg po in mice.

The discovery of novel 3-(pyrazin-2-yl)-1H-indazoles as potent pan-Pim kinase inhibitors.

The three Pim kinases are a small family of serine/threonine kinases regulating several signaling pathways that are fundamental to tumorigenesis. As such, the Pim kinases are a very attractive target for pharmacological inhibition in cancer therapy. Herein, we describe our efforts toward the development of a potent, pan-Pim inhibitor. The synthesis and hit-to-lead SAR development from a 3-(pyrazin-2-yl)-1H-indazole derived hit 2 to the identification of a series of potent, pan-Pim inhibitors such as 13o are described.

Discovery and Optimization of Quinazolinone-pyrrolopyrrolones as Potent and Orally Bioavailable Pan-Pim Kinase Inhibitors

The high expression of proviral insertion site of Moloney murine leukemia virus kinases (Pim-1, -2, and -3) in cancers, particularly the hematopoietic malignancies, is believed to play a role in promoting cell survival and proliferation while suppressing apoptosis. The three isoforms of Pim protein appear largely redundant in their oncogenic functions. Thus, a pan-Pim kinase inhibitor is highly desirable. However, cell active pan-Pim inhibitors have proven difficult to develop because Pim-2 has a low Km for ATP and therefore requires a very potent inhibitor to effectively block the kinase activity at cellular ATP concentrations. Herein, we report a series of quinazolinone-pyrrolopyrrolones as potent and selective pan-Pim inhibitors. In particular, compound 17 is orally efficacious in a mouse xenograft model (KMS-12 BM) of multiple myeloma, with 93% tumor growth inhibition at 50 mg/kg QD upon oral dosing.

Discovery and Optimization of Macrocyclic Quinoxaline-pyrrolo-dihydropiperidinones as Potent Pim-1/2 Kinase Inhibitors.

The identification of Pim-1/2 kinase overexpression in B-cell malignancies suggests that Pim kinase inhibitors will have utility in the treatment of lymphoma, leukemia, and multiple myeloma. Starting from a moderately potent quinoxaline-dihydropyrrolopiperidinone lead, we recognized the potential for macrocyclization and developed a series of 13-membered macrocycles. The structure-activity relationships of the macrocyclic linker were systematically explored, leading to the identification of 9c as a potent, subnanomolar inhibitor of Pim-1 and -2. This molecule also potently inhibited Pim kinase activity in KMS-12-BM, a multiple myeloma cell line with relatively high endogenous levels of Pim-1/2, both in vitro (pBAD IC50 = 25 nM) and in vivo (pBAD EC50 = 30 nM, unbound), and a 100 mg/kg daily dose was found to completely arrest the growth of KMS-12-BM xenografts in mice.

Discovery of imidazopyridazines as potent Pim-1/2 kinase inhibitors

High levels of Pim expression have been implicated in several hematopoietic and solid tumor cancers, suggesting that inhibition of Pim signaling could provide patients with therapeutic benefit. Herein, we describe our progress towards this goal using a screening hit (rac-1) as a starting point. Modification of the indazole ring resulted in the discovery of a series of imidazopyridazine-based Pim inhibitors exemplified by compound 22m, which was found to be a subnanomolar inhibitor of the Pim-1 and Pim-2 isoforms (IC50 values of 0.024nM and 0.095nM, respectively) and to potently inhibit the phosphorylation of BAD in a cell line that expresses high levels of all Pim isoforms, KMS-12-BM (IC50=28nM). Profiling of Pim-1 and Pim-2 expression levels in a panel of multiple myeloma cell lines and correlation of these data with the potency of compound 22m in a proliferation assay suggests that Pim-2 inhibition would be advantageous for this indication.

Abstract 5398: In vivo development of pan-Pim kinase small molecule inhibitors

Pim-1,-2, and -3 are constitutively active serine-threonine kinases which are partially redundant and regulate multiple pathways important for tumor growth and survival. One or more of the human Pims are over-expressed in multiple hematological tumor types (e.g. multiple myeloma (MM), NHL and AML) and in some solid tumors (e.g. prostate and SCLC). Pim over-expression correlates with malignancy and poor prognosis in several indications. Our goal was to generate a pan Pim kinase inhibitor with acceptable physical chemical properties and in vivo anti-tumor efficacy. Here we present data on two ATP-competitive, orally bioavailable pan Pim inhibitors, Compound I and Compound II. These inhibitors have potent enzymatic and cellular activity, acceptable pharmacokinetic properties (PK) and robust in vivo efficacy. In a kinase enzyme assay Compound I inhibits Pim-1 and Pim-2 activity with 0.4 nM and 0.7 nM IC50s, respectively, while Compound II is even more potent with Pim-1 and Pim-2 IC50s of 0.1 nM/0.1 nM. In a cellular assay which measures inhibition of the Pim downstream substrate phospho-BAD (p-BAD), compounds I and II demonstrate IC50s of 56 and 16 nM, respectively. In an in vivo pharmacodynamic assay (PD) to demonstrate on-target Pim activity, compounds I and II significantly inhibited p-BAD in KMS-12-BM multiple myeloma tumors for 16 hours post dose. Treatment of KMS-12-BM tumor xenografts with Compound I demonstrated robust in vivo anti-tumor efficacy resulting in 23% tumor regression at 50 mg/kg BID and tumor stasis at 100 mg/kg QD. Compound II demonstrated improved PK properties leading to greater anti-tumor efficacy of 33% tumor regression at 100 mg/kg QD and tumor stasis at 50 mg/kg QD. Compound II showed efficacy in an orthotopic model of multiple myeloma and in models of AML and DLBCL. Combination treatment of Compound II and the standard of care Dexamethasone in the multiple myeloma RPMI-8226 xenograft model demonstrated enhanced tumor growth inhibition compared to either single agent activity. In summary, Compound I and II are potent and selective inhibitors of Pim kinases with excellent in vivo properties. Pim kinase inhibitors, either as monotherapy or in combination with dexamethasone, may be effective clinical strategies for certain cancer patients. Citation Format: Bethany Mattson, Christine. E. Sastri, Nadia Guerrero, Dean Hickman, Jie Chen, Tian Wu, Hui-Ling Wang, Andrew Taskar, Brian Lanman, Anthony B. Reed, Jude Canon, J. Russell Lipford, Karen Rex. In vivo development of pan-Pim kinase small molecule inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5398. doi:10.1158/1538-7445.AM2015-5398

Abstract 494: Evaluation of soluble FLT3 ligand as a marker of bone marrow stress and recovery

Depletion of bone marrow (BM)-resident myeloid progenitor cells is a consequence of various anti-cancer treatments, including bispecific T-cell engager (BiTE®) antibodies targeting myeloid cell antigens. Currently, aspirates are required to determine the extent of myeloid cell depletion in the BM. This procedure is painful and potentially invasive for patients. As a surrogate marker for BM myeloid cell depletion, we evaluated soluble FLT3 ligand (FLT3L) in non-human primates treated with myeloid cell-depleting BiTE® antibodies. While FLT3L is not detectable in serum during steady-state hematopoiesis, it is detectable in response to hematopoietic deficiency. The goal of this study was to evaluate the relationship of serum FLT3L with depletion and recovery of BM myeloid cells in non-human primates treated with BiTE® antibodies against CD33. Serum FLT3L levels were determined using an anti-human FLT3L ELISA kit. Validation studies demonstrated that both the capture and detection antibodies recognized cynomolgus FLT3L. Serum FLT3L concentrations were determined using a standard curve with rhesus FLT3L (99.6% identical to cynomolgus FLT3L). Serum samples were taken from cynomolgus monkeys before, during and after treatment with anti-CD33 BiTE® antibodies and compared with the number of BM-resident target cells. In the first study, cynomolgus monkeys were dosed once with different dose levels of anti-CD33 BiTE® antibody ranging from 1- 15 μg/kg. BM-resident myeloid cells were transiently reduced in some, but not all, cohorts. Soluble FLT3L was detected only when there was a reduction in BM myeloid cells. When FLT3L was detectable, the serum concentration correlated with the extent of target cell reduction and FLT3L levels decreased as the myeloid cells recovered. In the second study, cynomolgus monkeys were dosed three times with anti-CD33 BiTE® antibody. In these studies target cell reduction was greater and persisted longer compared to the single dose studies. Similarly, the magnitude and duration of increased FLT3L in the multidose studies was also greater. Within the multi-dose study, FLT3L levels continued to increase as more BM-resident myeloid cells were depleted, suggesting that it was possible to detect cumulative stress to the BM. In summary, serum FLT3L levels are correlated with the extent and duration of myeloid cell depletion in the BM suggesting that serum FLT3L levels reflect depletion and subsequent myeloid cell replenishment. All studies to date have involved treatment with myeloid antigen-specific BiTE® antibodies; future studies will evaluate FLT3L levels following treatment with anti-lymphoid or solid tumor antigen-specific BiTE® antibodies to determine if FLT3L increases are associated with myeloid cell depletion or indicative of general BM stress or inflammation. The potential benefit of this marker is that it could allow for repeat non-invasive BM monitoring in patients on myelosuppressive therapies. Citation Format: Christine Sastri, Mercedesz Balazs, Priya Koppikar, James R. Lipford, Angela Coxon, Tara L. Arvedson. Evaluation of soluble FLT3 ligand as a marker of bone marrow stress and recovery. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 494.

Abstract 5396: Characterization of small molecule inhibitors of the PIM kinases in in vitro models of hematological malignancies

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The three members of the Pim kinase family, Pim-1, -2, and -3, are established oncogenes and are attractive targets in hematological malignancies. We have developed multiple potent and selective scaffolds of pan-Pim inhibitors with picomolar enzymatic potency and nanomolar cellular potency. Using these agents, we have observed that the abolishment of Pim activity impairs tumor cell viability in multiple settings, both in vitro and in vivo. We have developed numerous assays to measure Pim protein levels and activity, including Pim downstream markers p-PDCD4 and p-BAD, that might be broadly applicable to human tissues. These assays have allowed us to establish a correlation between Pim protein levels and sensitivity to Pim inhibition across multiple tumor settings, in vitro. We have observed that all tested multiple myeloma (MM) cell lines express high levels of Pim-2 protein and that pan-Pim inhibition impairs viability in 90% of these lines and induces apoptosis in a subset. In acute myelogenous leukemia (AML) cell lines, sensitivity to Pim inhibition significantly correlates with Pim-1 protein expression. Numerous diffuse, large B-cell lymphoma (DLBCL) cell lines have high Pim levels and many are sensitive to Pim inhibition. We have also assessed Pim expression and activity in human tumor and normal tissues. Studies performed with myeloma cells isolated from patient bone marrow aspirates have revealed elevated Pim-2 protein levels as well as sensitivity to ex vivo dosing with Pim inhibitors, as evidenced by inhibition of PDCD4 phosphorylation. Primary patient samples from numerous other hematological tumors have also been found to have high Pim-1 or Pim-2 protein levels. To expand the possible utility of Pim inhibitors in the clinic, we have combined our molecules with numerous clinical agents, including dexamethasone, carfilzomib, and PI3K inhibitors, across multiple settings, in vitro. In all indications surveyed, we have observed that the combination of Pim molecules and these agents can lead to synergistic effects on cell viability, apoptosis and pathway signaling. In some cases, cell lines that show mild or no response to either single agent alone are sensitive to combination treatment. Collectively, our data provide a rationale for the development of Pim kinase inhibitors for use either as monotherapy or in combination with other agents in diverse tumor settings. Citation Format: Christine E. Sastri, Nadia Guerrero, Dongyin Yu, Bethany Mattson, Ken Dellamaggiore, Yajing Yang, Paul Hughes, Hui-Ling Wang, Victor Cee, Brian A. Lanman, Liping Pettus, Anthony B. Reed, Bin Wu, Ryan Wurz, Andrew Tasker, Li-Ya Huang, Daniel Branstetter, Karen Rex, Jeffrey Winston, Teresa L. Burgess, Richard Kendall, J Russell Lipford. Characterization of small molecule inhibitors of the PIM kinases in in vitro models of hematological malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5396. doi:10.1158/1538-7445.AM2015-5396