Supplementary Tables 1-3 from Distinct Concentration-Dependent Effects of the Polo-like Kinase 1–Specific Inhibitor GSK461364A, Including Differential Effect on Apoptosis

Additions to Supplement Table 3 from Distinct Concentration-Dependent Effects of the Polo-like Kinase 1–Specific Inhibitor GSK461364A, Including Differential Effect on Apoptosis

We report the discovery of sulanemadlin (ALRN-6924), the first cell-permeating, stabilized α-helical peptide to enter clinical trials. ALRN-6924 is a "stapled peptide" that mimics the N-terminal domain of the p53 tumor suppressor protein. It binds with high affinity to both MDM2 and MDMX (also known as MDM4), the endogenous inhibitors of p53, to activate p53 signaling in cells having a non-mutant, or wild-type TP53 genotype (TP53-WT). Iterative structure-activity optimization endowed ALRN-6924 with favorable cell permeability, solubility, and pharmacokinetic and safety profiles. Intracellular proteolysis of ALRN-6924 forms a long-acting active metabolite with potent MDM2 and MDMX binding affinity and slow dissociation kinetics. At high doses, ALRN-6924 exhibits on-mechanism anticancer activity in TP53-WT tumor models. At lower doses, ALRN-6924 transiently arrests the cell cycle in healthy tissues to protect them from chemotherapy without protecting the TP53-mutant cancer cells. These results support the continued clinical evaluation of ALRN-6924 as an anticancer and chemoprotection agent.

Background: Myelosuppression is a common sequela of acute radiation injury due to sensitivity of proliferating bone marrow cells to ionizing radiation. ALRN-6924 is a clinical-stage, first-in-class, stabilized cell-permeating alpha-helical peptide drug that disrupts the interaction of the p53 tumor suppressor protein with its endogenous inhibitors, MDMX and MDM2, to induce transient, dose-dependent cell cycle arrest in p53-wild-type tissues. ALRN-6924 is being evaluated in clinical trials as a selective chemoprotection agent for patients with p53-mutant cancers to protect healthy normal cells from chemotherapy while not protecting p53-mutant cancer cells. We tested whether ALRN-6924 may similarly protect against radiation-induced toxicity in mouse models of acute radiation injury.

Abstract Aim: We investigated whether p53 activation with ALRN-6924 can prevent toxicity in mouse models of acute radiation injury. Materials and methods: ALRN-6924 is a clinical-stage, first-in-class, stabilized cell-permeating alpha-helical peptide that disrupts the interaction of the p53 tumor suppressor protein with its endogenous inhibitors, MDMX and MDM2. In previous experiments it was shown that ALRN-6924 can induce transient, dose-dependent cell cycle arrest in the bone marrow to protect those cells from chemotherapy while not protecting p53-mutant cancer cells. Because radiation (like chemotherapy) preferentially affects proliferating cells, we hypothesized that ALRN-6924 may also protect proliferating cells in normal tissues from radiation-induced cellular toxicity. Cell cycle arrest was measured in the bone marrow of ALRN-6924-treated C57BL/6 mice by flow cytometry using EdU incorporation. Serum levels of macrophage inhibitory cytokine-1 (MIC-1), a biomarker of p53 activation, were measured by ELISA. Cell proliferation and apoptosis were measured in formalin-fixed mouse bone marrow by immunohistochemistry analysis of Ki67 and cleaved PARP, respectively. C57BL/6 mice (n=7/group) were treated with one or more intravenous 2.4 mg/kg doses of ALRN-6924 at 24, 16, 8, or 1 hour (or combinations thereof) or placebo prior to an abdominally targeted (shielded body) 15 Gy radiation dose and then monitored for body weight (BW). Results: MIC-1 was elevated in the serum of ALRN-6924-treated mice in a dose-dependent fashion. Repeated doses of ALRN-6924 every 8 hrs yielded sustained MIC-1 elevation, which correlated with reduced Ki67 positivity in the bone marrow. Treatment-dependent changes in cPARP expression were evident, but minimal in magnitude. In a nonlethal radiation exposure model, ALRN-6924 yielded significant protection from radiation-induced BW loss in a schedule-dependent manner. Placebo-treated mice showed 10% to 15% BW loss five days after irradiation, while mice receiving one or more ALRN-6924 doses 8 hrs prior to irradiation had an average of 4% BW loss (p=0.008, two-sided t test). Conclusions: ALRN-6924 mitigates toxicity in a mouse model of acute radiation injury. The observed radioprotection effect correlates with pharmacodynamic markers of cell proliferation and cell cycle arrest after one or more doses of ALRN-6924, and further supports previous demonstrations of chemoprotection with ALRN-6924. These results provide a rationale to investigate ALRN-6924 as a radioprotective agent. Citation Format: Allen Annis, David Sutton, Manuel Aivado, Vojislav Vukovic. The investigational chemoprotection drug ALRN-6924, a dual inhibitor of MDMX and MDM2, shows potential for radioprotection [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P211.

BACKGROUND:MDM2/MDMX proteins are frequently elevated in hormone receptor-positive (ER+) breast cancer. We sought to determine the antitumor efficacy of the combination of ALRN-6924, a dual inhibitor of MDM2/MDMX, with chemotherapy in ER+ breast cancer models.METHODS:Three hundred two cell lines representing multiple tumor types were screened to confirm the role of TP53 status in ALRN-6924 efficacy. ER+ breast cancer cell lines (MCF-7 and ZR-75-1) were used to investigate the antitumor efficacy of ALRN-6924 combination. In vitro cell proliferation, cell cycle, and apoptosis assays were performed. Xenograft tumor volumes were measured, and reverse-phase protein array (RPPA), immunohistochemistry (IHC), and TUNEL assay of tumor tissues were performed to evaluate the in vivo pharmacodynamic effects of ALRN-6924 with paclitaxel.RESULTS:ALRN-6924 was active in wild-type TP53 (WT-TP53) cancer cell lines, but not mutant TP53. On ER+ breast cancer cell lines, it was synergistic in vitro and had enhanced in vivo antitumor activity with both paclitaxel and eribulin. Flow cytometry revealed signs of mitotic crisis in all treatment groups; however, S phase was only decreased in MCF-7 single agent and combinatorial ALRN-6924 arms. RPPA and IHC demonstrated an increase in p21 expression in both combinatorial and single agent ALRN-6924 in vivo treatment groups. Apoptotic assays revealed a significantly enhanced in vivo apoptotic rate in ALRN-6924 combined with paclitaxel treatment arm compared to either single agent.CONCLUSION:The significant synergy observed with ALRN-6924 in combination with chemotherapeutic agents supports further evaluation in patients with hormone receptor-positive breast cancer.

Abstract Aim: We investigated whether p53 activation with ALRN-6924 can prevent or reduce chemotherapy-induced hematopoietic toxicity while preserving or enhancing anti-tumor efficacy of chemotherapy in p53-mutant tumors. Materials and methods: ALRN-6924 is a clinical-stage, first-in-class, stabilized cell-permeating alpha-helical peptide that disrupts the interaction of the p53 tumor suppressor protein with its endogenous inhibitors, MDMX and MDM2. For p53 wild-type cells such as normal bone marrow, p53 activation can induce transient, dose-dependent cell cycle arrest, reducing sensitivity to chemotherapy-induced cellular toxicity. For p53-mutant cancer cells, ALRN-6924 has no effect on the cell cycle, leaving them vulnerable to chemotherapy. ALRN-6924-induced cell cycle arrest was measured by flow cytometry in human bone marrow CD34+ cells following incubation with ALRN-6924 ex vivo for 24 hours. DNA synthesis and DNA content were quantified by flow cytometry using EdU incorporation and Hoechst 33342 staining, respectively. Cell cycle arrest in the bone marrow of ALRN-6924-treated C57BL/6 mice was measured by flow cytometry using EdU incorporation in lineage negative, Kit positive hematopoietic stem and progenitor cells. Topotecan-induced DNA damage was measured in human bone marrow CD34+ cells by H2γX incorporation following exposure to vehicle or ALRN-6924 for 24 hours to induce cell cycle arrest, then incubated with topotecan for an additional 24 hours following a wash-out step. Topotecan-induced neutropenia was measured in female C57BL/6 mice following topotecan treatment on days 1-5 and either ALRN-6924 or vehicle on days 0-4. Female C57BL/6 mice bearing subcutaneous p53-mutant MC38 syngeneic tumors were treated with ALRN-6924, vehicle and topotecan on the same dosing regimen and followed until tumors reached >1000mm3. Results: ALRN-6924 induces transient, reversible cell cycle arrest in bone marrow cells in vitro and in vivo, and protects human bone marrow cells against topotecan-induced DNA damage ex vivo. In a mouse model of topotecan-induced neutropenia, ALRN-6924 protected against neutrophil depletion when daily administration started 24 hours prior to the 1st dose and 30 minutes before each subsequent dose of topotecan. ALRN-6924 does not diminish topotecan’s anti-tumor activity in the p53-mutant MC38 syngeneic mouse cancer model, with the ALRN-6924 + topotecan combination yielding modest enhancement of survival. Body weights and mortality data suggest ALRN-6924 and combinations with topotecan were tolerated at the doses tested. Conclusions: ALRN-6924 reduces chemotherapy-induced hematopoietic toxicity in healthy human bone marrow cells ex vivo and in mouse models of topotecan-induced neutropenia in vivo, while preserving or enhancing anti-tumor efficacy in p53-mutant tumors when administered intravenously prior to chemotherapy. These results support the first ALRN-6924 clinical trial for myelopreservation in topotecan-treated small-cell lung cancer patients (NCT04022876). Additional studies are underway to support ALRN-6924 as a tumor type-agnostic myelopreservation agent for cancer patients with tumors bearing p53 mutations who are treated with chemotherapy. Citation Format: Luis A Carvajal, David Sutton, Mariam Mounir, Joseph McClanaghan, Vincent Guerlavais, Manuel Aivado, Vojislav Vukovic, Allen Annis. The investigational peptide drug ALRN-6924, a dual inhibitor of MDMX and MDM2, is an effective myelopreservation agent [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C064. doi:10.1158/1535-7163.TARG-19-C064

Background: ALRN-6924, an α-helical stapled p53 peptide, is the first-in-class and only dual inhibitor of MDMX and MDM2 currently in clinical trials for both, solid and hematologic malignancies. In tumors with wild-type (WT) TP53, ALRN-6924 can induce p53-dependent cell cycle arrest and apoptosis and separately can invoke p53-mediated immunomodulatory effects, including but not limited to lymphocyte chemotaxis and diminution of PD-L1 expression on tumor cells. This report begins to describe the biologic underpinnings of PsP in selected PTCL patients (pts) treated with ALRN-6924. Despite imaging-based progressive disease (PD), careful monitoring for clinical benefit in light of conflicting imaging (PsP) results led to the decision to treat beyond radiologic progression and the observation of subsequent, delayed radiologic responses.

Xenotransplantation of human cancers into immunodeficient mice is a very useful approach for studying human tumor biology. However, the occasional occurrence of lymphomagenesis in some mice can spoil the model and must be investigated in detail. We found that a high percentage (32.5%, 26/80) of cancer patient-derived xenografts (PDXs) resembled lymphoma in NOD/SCID mice. Of the 26 xenografts, 23 were human-derived expressing human CD45 (hCD45+) and proved to be of the B-cell subtype (CD3-/CD20+), and they were all positive for Epstein - Barr virus (EBV). The remaining 3 xenografts proved to be mouse-derived for both hCD45-and negative amplification of a human gene. The most interesting finding is that gastric cancer had much higher rates (24/126, 19.0%) of lymphoma formation in the PDX model than did colorectal cancer (1/43, 2.3%). Statistical analysis revealed that cancer type and inflammation in the parent tumor are significantly associated with lymphomagenesis. Further validation discovered lymphomagenesis by inoculating only gastritis mucosa. Therefore, our findings suggest that it is necessary to take precautions when directly xenografting cancer tissues with remarkable baseline inflammation, such as gastric cancer into immunodeficient NOD/SCID strains. Further, the established xenograft models should be validated by both leukocyte markers and human gene signatures.

Abstract Oncogenic BRAF, which drives cell transformation and proliferation, has been detected in approximately 50% of human malignant melanomas and 5% to 15% of colorectal cancers. Despite the remarkable clinical activities achieved by vemurafenib and dabrafenib in treating BRAFV600E metastatic melanoma, their clinical efficacy in BRAFV600E colorectal cancer is far less impressive. Prior studies suggested that feedback activation of EGFR and MAPK signaling upon BRAF inhibition might contribute to the relative unresponsiveness of colorectal cancer to the first-generation BRAF inhibitors. Here, we report characterization of a dual RAF kinase/EGFR inhibitor, BGB-283, which is currently under clinical investigation. In vitro, BGB-283 potently inhibits BRAFV600E-activated ERK phosphorylation and cell proliferation. It demonstrates selective cytotoxicity and preferentially inhibits proliferation of cancer cells harboring BRAFV600E and EGFR mutation/amplification. In BRAFV600E colorectal cancer cell lines, BGB-283 effectively inhibits the reactivation of EGFR and EGFR-mediated cell proliferation. In vivo, BGB-283 treatment leads to dose-dependent tumor growth inhibition accompanied by partial and complete tumor regressions in both cell line-derived and primary human colorectal tumor xenografts bearing BRAFV600E mutation. These findings support BGB-283 as a potent antitumor drug candidate with clinical potential for treating colorectal cancer harboring BRAFV600E mutation. Mol Cancer Ther; 14(10); 2187–97. ©2015 AACR.

Abstract Purpose: Despite their preclinical promise, previous MEK inhibitors have shown little benefit for patients. This likely reflects the narrow therapeutic window for MEK inhibitors due to the essential role of the P42/44 MAPK pathway in many nontumor tissues. GSK1120212 is a potent and selective allosteric inhibitor of the MEK1 and MEK2 (MEK1/2) enzymes with promising antitumor activity in a phase I clinical trial (ASCO 2010). Our studies characterize GSK1120212' enzymatic, cellular, and in vivo activities, describing its unusually long circulating half-life. Experimental Design: Enzymatic studies were conducted to determine GSK1120212 inhibition of recombinant MEK, following or preceding RAF kinase activation. Cellular studies examined GSK1120212 inhibition of ERK1 and 2 phosphorylation (p-ERK1/2) as well as MEK1/2 phosphorylation and activation. Further studies explored the sensitivity of cancer cell lines, and drug pharmacokinetics and efficacy in multiple tumor xenograft models. Results: In enzymatic and cellular studies, GSK1120212 inhibits MEK1/2 kinase activity and prevents Raf-dependent MEK phosphorylation (S217 for MEK1), producing prolonged p-ERK1/2 inhibition. Potent cell growth inhibition was evident in most tumor lines with mutant BRAF or Ras. In xenografted tumor models, GSK1120212 orally dosed once daily had a long circulating half-life and sustained suppression of p-ERK1/2 for more than 24 hours; GSK1120212 also reduced tumor Ki67, increased p27Kip1/CDKN1B, and caused tumor growth inhibition in multiple tumor models. The largest antitumor effect was among tumors harboring mutant BRAF or Ras. Conclusions: GSK1120212 combines high potency, selectivity, and long circulating half-life, offering promise for successfully targeting the narrow therapeutic window anticipated for clinical MEK inhibitors. Clin Cancer Res; 17(5); 989–1000. ©2011 AACR.

Phosphoinositide 3-kinase α (PI3Kα) is a critical regulator of cell growth and transformation, and its signaling pathway is the most commonly mutated pathway in human cancers. The mammalian target of rapamycin (mTOR), a class IV PI3K protein kinase, is also a central regulator of cell growth, and mTOR inhibitors are believed to augment the antiproliferative efficacy of PI3K/AKT pathway inhibition. 2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide (GSK2126458, 1) has been identified as a highly potent, orally bioavailable inhibitor of PI3Kα and mTOR with in vivo activity in both pharmacodynamic and tumor growth efficacy models. Compound 1 is currently being evaluated in human clinical trials for the treatment of cancer.

Centromere-associated protein-E (CENP-E) is a kinetochore-associated mitotic kinesin that is thought to function as the key receptor responsible for mitotic checkpoint signal transduction after interaction with spindle microtubules. We have identified GSK923295, an allosteric inhibitor of CENP-E kinesin motor ATPase activity, and mapped the inhibitor binding site to a region similar to that bound by loop-5 inhibitors of the kinesin KSP/Eg5. Unlike these KSP inhibitors, which block release of ADP and destabilize motor-microtubule interaction, GSK923295 inhibited release of inorganic phosphate and stabilized CENP-E motor domain interaction with microtubules. Inhibition of CENP-E motor activity in cultured cells and tumor xenografts caused failure of metaphase chromosome alignment and induced mitotic arrest, indicating that tight binding of CENP-E to microtubules is insufficient to satisfy the mitotic checkpoint. Consistent with genetic studies in mice suggesting that decreased CENP-E function can have a tumor-suppressive effect, inhibition of CENP-E induced tumor cell apoptosis and tumor regression.

The Aurora kinases play critical roles in the regulation of mitosis and are frequently overexpressed or amplified in human tumors. Selective inhibitors may provide a new therapy for the treatment of tumors with Aurora kinase amplification. Herein we describe our lead optimization efforts within a 7-azaindole-based series culminating in the identification of GSK1070916 (17k). Key to the advancement of the series was the introduction of a 2-aryl group containing a basic amine onto the azaindole leading to significantly improved cellular activity. Compound 17k is a potent and selective ATP-competitive inhibitor of Aurora B and C with K(i)* values of 0.38 +/- 0.29 and 1.5 +/- 0.4 nM, respectively, and is >250-fold selective over Aurora A. Biochemical characterization revealed that compound 17k has an extremely slow dissociation half-life from Aurora B (>480 min), distinguishing it from clinical compounds 1 and 2. In vitro treatment of A549 human lung cancer cells with compound 17k results in a potent antiproliferative effect (EC(50) = 7 nM). Intraperitoneal administration of 17k in mice bearing human tumor xenografts leads to inhibition of histone H3 phosphorylation at serine 10 in human colon cancer (Colo205) and tumor regression in human leukemia (HL-60). Compound 17k is being progressed to human clinical trials.

Inhibition of mitotic kinesins represents a novel approach for the discovery of a new generation of anti-mitotic cancer chemotherapeutics. We report here the discovery of the first potent and selective inhibitor of centromere-associated protein E (CENP-E) 3-chloro-N-{(1S)-2-[(N,N-dimethylglycyl)amino]-1-[(4-{8-[(1S)-1-hydroxyethyl]imidazo[1,2-a]pyridin-2-yl}phenyl)methyl]ethyl}-4-[(1-methylethyl)oxy]benzamide (GSK923295; 1), starting from a high-throughput screening hit, 3-chloro-4-isopropoxybenzoic acid 2. Compound 1 has demonstrated broad antitumor activity in vivo and is currently in human clinical trials.

Abstract Polo-like kinase 1 (Plk1) is a conserved serine/threonine kinase that plays an essential role in regulating the many processes involved in mitotic entry and progression. In humans, Plk1 is expressed primarily during late G2 and M phases and, in conjunction with Cdk1/cyclin B1, acts as master regulatory kinases for the myriad protein substrates involved in mitosis. Plk1 overexpression is strongly associated with cancer and has been correlated with poor prognosis in a broad range of human tumor types. We have identified a potent, selective, reversible, ATP-competitive inhibitor of Plk1, GSK461364A, capable of inhibiting cell growth of most proliferating cancer cell lines tested. We observe distinct cell cycle effects of GSK461364A depending on the dose used. The predominant phenotype for cells treated with GSK461364A is prometaphase arrest with characteristic collapsed polar polo spindle. At high concentrations, GSK461364A delays mitotic entry in G2 followed by gradual progression into terminal mitosis; in some cell lines, this correlates with decreased apoptosis. Cell culture growth inhibition by GSK461364A can be cytostatic or cytotoxic but leads to tumor regression in xenograft tumor models under proper dose scheduling. Finally, we describe pharmacodynamic biomarkers of GSK461364A activity (pHH3 and Plk1) that are currently being evaluated in human cancer clinical trials. [Cancer Res 2009;69(17):6969–77]

The phosphoinositide 3‐kinase (PI3K) pathway is among the most commonly activated pathways in human cancer. The biological role of PI3K in growth and survival of cancer cells and the prevalence of activating mutations in human cancers are well documented and a significant proportion of tumors would be predicted to benefit from inhibition of this pathway. Here we report on the characterization of the pan‐PI3K pyridylsulfonamide inhibitor GSK2126458: a very potent (PI3K app K i = 19 pM), reversible, ATP‐competitive inhibitor of wild‐type PI3K and the ‘hotspot’ activating mutants of p110 found in human cancer. GSK2126458 demonstrated good selectivity for the PI3K family of enzymes including the mTORC1 and mTORC2 complexes, when evaluated in a large panel of protein kinases. Consistent with potent PI3K enzyme inhibition, GSK2126458 decreased the cellular levels of phosphorylated AKT, p70 S6K , PRAS40 and ERK in a concentration and time dependent manner, the IC50 for pAKT in the HCC1954 breast carcinoma cell line was 2 nM. GSK2126458 induced the nuclear translocation of the FOXO3a transcription factor in a concentration dependent manner that mechanistically appeared bimodal. Growth inhibition was time and concentration dependent with a 3 day exposure resulting in a growth IC 50 (gIC 50 ) of 9 nM in HCC1954 cells with evidence of cell death. Cell death correlated with induction of caspase 3 and 7 activity suggesting apoptosis was the mechanism of cell death. GSK2126458 had a breadth of activity for potent cell growth inhibition and induction of cell death in a variety of human cancer cells. GSK2126458 demonstrated robust, dose dependent in vivo pharmacodynamic activity as measured by inhibition of phospho‐AKT in advanced BT474 breast cancer cell xenograft tumors. Inhibition of AKT phosphorylation was rapid and lasted for several hours after a single oral administration of GSK2126458. Transient increases in plasma insulin and blood glucose were observed. Evaluation of in vivo tumor growth effects of GSK2126458 in xenograft models demonstrated dose dependent tumor growth delay in several models of diverse tumor lineage and tumor regression in a breast cancer xenograft. The biological profile of GSK2126458 supports the clinical advancement of this compound and GSK2126458 has entered Phase I human clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C63.

The protein kinases, Aurora A, B, and C have critical roles in the regulation of mitosis and are frequently overexpressed or amplified in human tumors. GSK1070916, is a novel ATP competitive inhibitor that is highly potent and selective for Aurora B/C kinases. Human tumor cells treated with GSK1070916 show dose-dependent inhibition of phosphorylation on serine 10 of Histone H3, a substrate specific for Aurora B kinase. Moreover, GSK1070916 inhibits the proliferation of tumor cells with EC(50) values of <10 nmol/L in over 100 cell lines spanning a broad range of tumor types. Although GSK1070916 has potent activity against proliferating cells, a dramatic shift in potency is observed in primary, nondividing, normal human vein endothelial cells, consistent with the proposed mechanism. We further determined that treated cells do not arrest in mitosis but instead fail to divide and become polyploid, ultimately leading to apoptosis. GSK1070916 shows dose-dependent inhibition of phosphorylation of an Aurora B-specific substrate in mice and consistent with its broad cellular activity, has antitumor effects in 10 human tumor xenograft models including breast, colon, lung, and two leukemia models. These results show that GSK1070916 is a potent Aurora B/C kinase inhibitor that has the potential for antitumor activity in a wide range of human cancers.