Patent Description:
The application also provides Onvansertib for use in a method of treating cancer in a patient, the method comprising: (i) determining the phosphorylation of serine <NUM> of TCTP in a sample obtained from the patient; and (ii) treating the patient with onvansertib if the phosphorylation is decreased after the cells or cell-free TCTP have been exposed to onvansertib compared to before they have been exposed to onvansertib, wherein the cancer is acute myeloid leukemia (AML).

Polo-like kinase <NUM> (PLK1) is the most well characterized member of the <NUM> members of the family of serine/threonine protein kinases and strongly promotes the progression of cells through mitosis. PLK1 performs several important functions throughout mitotic (M) phase of the cell cycle, including the regulation of centrosome maturation and spindle assembly, the removal of cohesins from chromosome arms, the inactivation of anaphase-promoting complex/cyclosome (APC/C) inhibitors, and the regulation of mitotic exit and cytokinesis (Stebhardt, <NUM>). It plays a key role in centrosome functions and the assembly of bipolar spindles. It also acts as a negative regulator of p53 family members leading to ubiquitination and subsequent degradation of p53/TP53, inhibition of the p73/TP73 mediated pro-apoptotic functions and phosphorylation/degradation of bora, a cofactor of Aurora kinase A. During the various stages of mitosis PLK1 localizes to the centrosomes, kinetochores and central spindle. PLK1 is aberrantly overexpressed in a variety of human cancers including acute myeloid leukemia (AML), breast, ovarian, non-small cell lung, colon, head and neck, endometrial and esophageal carcinomas and is correlated with cellular proliferation and poor prognosis (Degenhardt, <NUM>; Liu et al. , <NUM>; Cancer Genome Atlas).

Onvansertib (also known as PCM-<NUM>, NMS-<NUM>, NMS-<NUM>, "compound of formula (<NUM>)" in <CIT>; IUPAC name <NUM>-(<NUM>-hydroxyethyl)-<NUM>-{[<NUM>-(<NUM>-methylpiperazin-<NUM>-yl)-<NUM>-(trifluoromethoxy) phenyl] amino}- <NUM>,<NUM>-dihydro-<NUM>-pyrazolo[<NUM>,<NUM>-h] quinazoline-<NUM>-carboxamide) is the first PLK1 specific ATP competitive inhibitor administered by oral route to enter clinical trials with proven antitumor activity in different preclinical models (Beria et al. , <NUM>; Hartsink-Segers et al. , <NUM>; Sero et al. , <NUM>; Valsasina et al. , <NUM>; Casolaro et al. The compound shows high potency in proliferation assays having low nanomolar activity on a large number of cell lines, both from solid as well as hematologic tumors. Onvansertib potently causes a mitotic cell-cycle arrest followed by apoptosis in cancer cell lines and inhibits xenograft tumor growth with a clear PLK1-related mechanism of action at well tolerated doses in mice after oral administration. In addition, onvansertib shows activity in combination therapy with approved cytotoxic drugs, such as irinotecan, in which there is enhanced tumor regression in HT29 human colon adenocarcinoma xenografts compared to each agent alone (Valsasina et al. , <NUM>; see also <CIT>), and shows prolonged survival of animals in a disseminated model of AML in combination therapy with cytarabine (Valsasina et al. , <NUM>; Casolaro et al. Onvansertib has favorable pharmacologic parameters and good oral bioavailability in rodent and nonrodent species, as well as proven antitumor activity in different nonclinical models using a variety of dosing regimens, which may potentially provide a high degree of flexibility in dosing schedules, warranting investigation in clinical settings. Onvansertib has several advantages over previous PLK inhibitors, including high selectivity for PLK1 only, oral availability and half-life of ~<NUM> hours.

A Phase <NUM> dose-escalation study with onvansertib has been conducted in adult subjects with advanced/metastatic solid tumors at a single study site in the US. The primary objective of that study was to determine a maximum tolerated dose (MTD) of onvansertib in adult subjects with advanced/metastatic solid tumors. Secondary objectives of the study were to define antitumor activity. In that study, a recommended phase <NUM> dose of <NUM>/m<NUM> was established and <NUM> of <NUM> evaluable patients had stable disease.

Based on the above, there is a need for a method that enriches for the subset of subjects that have a greater likelihood of responding to onvansertib or other PLK1 inhibitors. The present invention satisfies that need by providing methods that predict the efficacy of a PLK1 inhibitor on a cancer by determining the extent of inhibition of phosphorylation of a PLK1 target by the PLK1 inhibitor.

<NPL>, presents preliminary patient data from a phase 1b/<NUM> trial and describes biomarker assays that will be used to evaluate target engagement of PCM-<NUM> in AML patients.

Provided are methods of selecting cancer patients for treatment with onvansertib comprising.

Also provided is Onvansertib for use in methods of treating cancer in a patient, the methods comprising:.

wherein the cancer is acute myeloid leukemia (AML).

Additionally, the use of "or" is intended to include "and/or", unless the context clearly indicates otherwise.

The present invention is based in part on the discovery that the effectiveness of a PLK1 inhibitor in treating a cancer in a patient can be quickly and easily determined by determining whether the PLK1 inhibitor is able to inhibit PLK1 activity in cells of the cancer. That discovery is established by the studies described in Examples below. Those Examples establish that inhibition of phosphorylation of a PLK1 target (TCTP) from a cancer (acute myeloid leukemia) in a patient by a PLK1 inhibitor (onvansertib) identifies a cancer that responds to treatment of the patient with the PLK1 inhibitor. With those results, the skilled artisan would understand that a measurement of inhibition of phosphorylation of any PLK1 target in any cancer in any patient by any PLK1 inhibitor would identify a cancer that responds to treatment with that PLK1 inhibitor.

Thus, the present invention is directed to a method of selecting cancer patients for treatment with onvansertib comprising:.

Phosphorylation of any PLK1 target may be measured. Nonlimiting examples of PLK1 phosphorylation targets that can be evaluated for reduced phosphorylation include TRF1, Mre11, PTP1B, Orc2, Hbo1, BubR1, WDR62, IRS2, LSD1, caspase-<NUM>, NudC, PTEN, BORA, BUB1B/BUBR1, CCNB1, CDC25C, CEP55, ECT2, ERCC6L, FBXO5/EMI1, FOXM1, KIF20A/MKLP2, CENPU, NEDD1, NINL, NPM1, NUDC, PKMYT1/MYT1, KIZ, PPP1R12A/MYPT1, PRC1, RACGAP1/CYK4, SGO1, STAG2/SA2, TEX14, TOPORS, p73/TP73, WEE1, HNRNPU and translational control tumor protein (TCTP). In the present invention the PLK1 target that is evaluated for phosphorylation inhibition is TCTP (UniProtKB P13693). PLK1 phosphorylates TCTP at serine <NUM> and threonine <NUM> (Cucchi et al, <NUM>; Acunzo et al.

In some embodiments, the patient is treated with the PLK1 inhibitor and a sample of the cancer is taken before and after that treatment, and phosphorylation of the target is measured in those samples, to determine whether the PLK1 inhibitor is effective. In other embodiments, cancer cells (e.g., in blood, lymph, bone marrow or tissue such as biopsy tissue or cells from a needle aspiration) are removed from the patient's body, and those cells are treated with the PLK1 inhibitor in vitro. Such samples can be any tissue or bodily fluid where the cancer is present.

The cancer cells that are evaluated in these methods can be in tissue or in a liquid of the patient, e.g., any liquid or tissue harboring any of the cancers identified below, for example, the blood, a tissue aspirate, bone marrow, urine etc. of the patient. In some embodiments where the cancer is in the blood of the patient, the cells to be utilized to evaluate PLK1 inhibition can be taken in a sample of the patient's lymph, bone marrow or blood. Any cancer in the blood of a patient can be evaluated by these methods, for example, the cancer may be acute myeloid leukemia (AML), B-cell lymphoma, or from a metastatic tumor. In the present invetion, the cancer is acute myeloid leukemia (AML). Methods to evaluate cells from metastatic tumors in blood are known in the art. See, e.g., Racila et al.

These methods are not narrowly limited to any particular time after treatment with the PLK1 inhibitor that the sample is taken and evaluated for inhibition of phosphorylation of the PLK1 target. The determination of the time interval after treatment that the sample is taken for any PLK1 target and PLK1 inhibitor treatment can be made without undue experimentation. Where the PLK1 inhibitor is onvansertib (oral administration, half-life about <NUM> hours) and the PLK1 target is TCTP, it is believed that the sample can be taken and evaluated anytime from <NUM> to <NUM> hours after treatment. In some embodiments, the phosphorylation of the PLK1 target is determined at least two hours after treatment. In other embodiments, the phosphorylation of the PLK1 target is determined at least about three hours after treatment.

This method may be utilized in cells of any cancer. Exemplary cancers include, but are not limited to, acute lymphoblastic leukemia, AML, adrenocortical carcinoma, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), extrahepatic bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas/carcinoids, carcinoid tumor, gastrointestinal, central nervous system lymphoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, mycosis fungoides, Sezary Syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor glioma, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors (endocrine pancreas), Kaposi Sarcoma, kidney (renal cell) cancer, kidney cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, Waldenstrom macroglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, mesothelioma malignant, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing family of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterine sarcoma, skin cancer (nonmelanoma), skin cancer (melanoma), Merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, gestational trophoblastic tumor, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms Tumor.

The cancer may have elevated PLK1 activity when compared to non-cancerous cells. Nonlimiting examples of such cancers include ovarian cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, squamous cell carcinoma, a hematopoietic tumor of lymphoid lineage, a hematopoietic tumor of myeloid lineage, a tumor of the central or peripheral nervous system, acute myeloid leukemia, B-cell lymphoma, adrenocortical, esophageal, stomach, and head and neck cancer.

In the present invention, the cancer is acute myeloid leukemia (AML).

In some embodiments, after the first determination prior to treatment with the PLK inhibitor, the determined PLK1 activity is compared with normal, i.e., non-cancerous, PLK1 activity, and the patient or cancer sample is not treated with the PLK1 inhibitor unless PLK1 activity is elevated in the cancer. The rationale for these embodiments is that PLK1 inhibitor treatment is most likely to benefit patients with cancers that have elevated PLK1 activity. In some embodiments, the patient or cancer sample is not treated with the PLK1 inhibitor if PLK1 activity is below normal.

As used herein, "normal" PLK1 activity is PLK1 activity in non-cancerous cells, either from the patient or average in a population, preferably in tissues or cells that are matched or similar to the cancerous tissue.

Any PLK1 inhibitor may be evaluated using these methods. Nonlimiting examples include onvansertib, BI2536, volasertib (BI <NUM>), GSK461364, HMN-<NUM>, HMN-<NUM>, AZD1775, CYC140, rigosertib (ON-<NUM>), MLN0905, TKM-<NUM>, TAK-<NUM> or Ro3280.

In the present invention, the PLK1 inhibitor is onvansertib. In the present invention, the cancer is acute myeloid leukemia (AML).

The phosphorylation of the PLK1 target (TCTP) can be determined by any method known in the art. In some embodiments, the phosphorylation is determined immunochemically, e.g., using antibodies that (a) bind to the unphosphorylated target and (b) bind to the phosphorylated, but not the unphosphorylated target. These methods are not narrowly limited to any particular immunochemical method. Examples of methods that can be utilized include western blot (as in Example <NUM> below), dot blot, ELISA, immunohistochemistry, and immuno-PCR (for pTCTP detection).

Immuno-PCR is analogous to ELISA, except that an oligonucleotide is the signal-generating moiety rather than an enzyme. The signal from the oligonucleotide is generated by polymerase chain reaction (PCR) amplification. Because PCR amplifies the oligonucleotide many fold, normally resulting in a dye-labeled amplification product, immuno-PCR is extremely sensitive, and, when used in the present methods, is able to detect a very small amount of phosphorylated TCTP.

Since immuno-PCR is analogous to ELISA, immuno-PCR can be utilized in any format for detecting pTCTP that has been described to detect other proteins with ELISA, for example direct, indirect, sandwich or competitive formats, using any solid phase (e.g., microtiter plates, beads, etc.) known in the art (see, e.g., BioRad, <NUM>).

A diagram of a non-limiting example of immuno-PCR for detecting pTCTP is provided in <FIG>. In that example, avidin is covalently or noncovalently bound to a solid phase. A biotin-labeled TCTP capture antibody, which binds to a first epitope ("EPITOPE-<NUM>") on phosphorylated TCTP ("pTCTP") is bound to the avidin. A sample suspected of having pTCTP is added, then a TCTP reporter antibody is then added. In this assay, either the capture antibody or the reporter antibody can bind to pTCTP but not to unphosphorylated TCTP. In some embodiments, the reporter antibody can bind to pTCTP but not to unphosphorylated TCTP. A double stranded DNA molecule (dsDNA) is on the reporter antibody. PCR to amplify the dsDNA is then conducted. There will be a PCR product only if the sample had pTCTP; the amount of that PCR product is proportional to the amount of pTCTP in the sample. The PCR product can then be quantified to determine the amount of pTCTP that is present in the sample.

In any of the above methods, phosphorylation of any amino acid residue of the target protein can be measured. For example, with the target TCTP, phosphorylation of serine <NUM> or threonine <NUM>, or both, can be measured. In the present invention, phosphorylation of serine <NUM> is measured.

These methods can be utilized to determine the effectiveness of the PLK1 inhibitor on the cancer. This is demonstrated in Example <NUM> below, where the PLK1 inhibitor onvansertib inhibited phosphorylation in the PLK1 target TCTP in AML cells in patients that responded to onvansertib but not in AML cells in patients that did not respond to onvansertib.

The present invention also encompasses certain variations of the above methods. For example, rather than evaluating PLK1 inhibition in cells of the cancer, cell-free PLK1 and/or the PLK1 target (e.g., TCTP) in patient serum can be evaluated for inhibition of target phosphorylation when exposed to the PLK1 inhibitor. For this variation, a very sensitive method, for example immuno-PCR, may be utilized to determine inhibition of phosphorylation.

In an additional variation, the ability of a PLK1 inhibitor to inhibit PLK1 in a cancer can be determined by determining the ability of the PLK1 inhibitor to inhibit PLK1 activity in the cancer using the methods described herein.

As demonstrated in the Examples below, the inhibition of phosphorylation of a PLK1 target by a PLK1 inhibitor indicates that the PLK1 inhibitor is effective against the cancer. These methods can therefore be used to make an early determination of whether the PLK1 inhibitor is effective.

The determination of the percentage of inhibition of PLK1 target phosphorylation that indicates effectiveness of a particular PLK1 inhibitor can be made without undue experimentation and depends on the PLK1 target, the PLK1 inhibitor, the time after treatment that a sample is taken for testing, how effectiveness of treatment is measured, and on the relative number of false positives or false negatives that are desired. For example, in the Examples below, when the percent inhibition was set at <NUM>%, there was one false negative (starred bar in <FIG>), where the inhibition of pTCTP was only <NUM>%, but the PLK1 inhibitor treatment caused a large reduction in % bone marrow blast and an objective response in that patient. With a percentage inhibition set at <NUM>%, there would be no false negatives. The percent inhibition that indicates effectiveness of PLK1 inhibitor treatment can thus be set at any percentage, e.g., <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>%, or any other percentage.

Thus, in some embodiments, a reduction of at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% in the phosphorylation of the PLK1 target after treatment indicates that the treatment is effective.

The above methods can be utilized to identify candidates in a trial of the efficacy of the PLK1 inhibitor against the cancer. In some embodiments of that application, the patient is not eligible for participation in the trial if the phosphorylation of the PLK1 target is not reduced at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% after the treatment relative to the phosphorylation of TCTP before the treatment.

The determination of the effectiveness of the PLK1 inhibitor treatment described above can be utilized to decide whether to therapeutically treat the patient with the PLK1 inhibitor, where the patient is only treated with the PLK1 inhibitor if the percentage of reduction of phosphorylation of the PLK1 target is above the threshold set as described above. Thus, in some embodiments, the patient is therapeutically treated with the PLK1 inhibitor only if the phosphorylation of TCTP is reduced at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% after the treatment relative to the phosphorylation of TCTP before the treatment.

These methods can be used when the PLK1 inhibitor is used alone in treating the cancer, or when the patient is also being treated, or being considered for treatment, of the cancer with a PLK1 inhibitor in combination with one or more anti-neoplastic agent that is not a PLK1 inhibitor. Non-limiting examples of such anti-cancer agents are cisplatin, cytarabine, decitabine, doxorubicin, gemcitabine, paclitaxel, SN38, sorafenib, velcade, abiraterone, ibrutinib, acalabrutinib, azacitidine, venetoclax, CPT11, 5FU, bevacizumab, bortezomib, a histone deacetylase inhibitor. In various embodiments, the patient continues treatment with the anti-cancer agent whether or not the patient is therapeutically treated with the PLK1 inhibitor.

In some embodiments, the cancer is acute myeloid leukemia (AML) and the patient is also being treated, or being considered for treatment (alone or in combination with a PLK1 inhibitor), with cytarabine, azacitidine, venetoclax, decitabine, a FLT3 inhibitor, or a combination thereof.

Preferred embodiments are described in the following examples.

This study is directed to the determination of whether the inhibition of phosphorylation of TCTP in cells of AML in patient blood by an inhibitor of PLK1, onvansertib, is predictive of the efficacy of onvansertib treatment for AML. The data establishes that onvansertib inhibition of TCTP phosphorylation in AML cells does predict response of AML to onvansertib treatment.

Patient data provided herein is from patients in clinical trial NCT03303339, Onvansertib in Combination With Either Low-dose Cytarabine (LDAC) or Decitabine in Adult Patients With Acute Myeloid Leukemia (AML) (ClinicalTrials.

Determination of phosphorylation of serine <NUM> of TCTP in blood was as follows. Peripheral blood mononuclear cells (PBMC) were isolated from a Cellsave blood tube (Menarini Silicon Biosystems) using Leucosep centrifuge tubes (VWR) and Histopaque-<NUM> (Sigma) according to the manufacturer's recommendations.

Protein extracts were prepared from PBMC and cell lines using M-PER buffer (ThermoFisher) with <NUM> NaCl and 1x protease and phosphatase inhibitors cocktails (ThermoFisher). Protein concentration was measured with the Pierce BCA protein assay kit (ThermoFisher).

Western blots were performed as Simple Western™ assays using the Wes system (ProteinSimple), a combination of capillary electrophoresis and immunodetection techniques, following the manufacturer's protocols. Briefly, extracts with equal protein concentration were mixed with <NUM>× sample buffer and <NUM>× fluorescent master mix. Denatured protein samples, biotinylated ladder (ProteinSimple), primary antibodies, horseradish peroxidase (HRP)-conjugated secondary antibody (ProteinSimple), chemiluminescence substrate and wash buffer were dispensed into respective wells of the assay plate and placed in Wes equipment. Primary antibodies were purchased from Cell Signaling Technology: phospho-TCTP-Ser46 (#<NUM>) and TCTP (#<NUM>), and used at a concentration of <NUM>:<NUM>. Quantitative analysis was performed using Compass software (ProteinSimple). Signal intensity (area) of pTCTP was normalized to the peak area of TCTP and reported as %pTCTP.

PLK1 inhibition in patients was assessed in blood taken <NUM>-hours following administration of onvansertib at peak concentration (Cmax).

Responders were defined as patients showing a decrease in circulating and bone marrow blasts during treatment.

Three of <NUM> patients treated with onvansertib in combination with low-dose cytarabine (LDAC) or decitabine exhibited a response to the treatment, as shown by decreases in the percentage of leukemic cells in blood (<FIG>) and bone marrow (<FIG>), with <NUM> patients (<NUM>-<NUM> and <NUM>-<NUM>) having decreases in bone marrow from <NUM>% to <NUM>% and <NUM>% to <NUM>%, respectively.

If effective in inhibiting PLK1 in vivo, onvansertib should prevent TCTP phosphorylation, as illustrated in <FIG>, particularly since onvansertib inhibits pTCTP in vitro, in a dose-dependent manner (<FIG>).

AML cell lines MV4-<NUM> and HL-<NUM> were treated for <NUM> minutes with onvansertib (PCM-<NUM>)(<NUM> or <NUM>), cytarabine (<NUM> or <NUM>) or decitabine (<NUM> or <NUM>). Proteins were then extracted from the cells and assessed for pTCTP. As shown in <FIG>, onvansertib, but not cytarabine or decitabine, inhibited pTCTP.

<FIG> shows percentage of leukemic cells in blood in three patients that were administered onvansertib and LDAC (cytarabine). Patient <NUM>-<NUM> responded to the treatment, and the other two patients did not. As shown in <FIG>, only the responder showed a decrease in pTCTP, as early as three hours from the initiation of treatment.

<FIG> shows percentage of leukemic cells in blood in three patients that were administered onvansertib and decitabine. Patients <NUM>-<NUM> and <NUM>-<NUM> both responded to the treatment, and patient <NUM>-<NUM> did not. As shown in <FIG>, only the two responders showed a decrease in pTCTP, both as early as three hours.

The data in <FIG> and <FIG> show that responders can be identified within <NUM> days of starting treatment using the TCTP phosphorylation method described herein, while responders could not be identified until <NUM> days or longer by determining % leukemic cells in blood.

<FIG> shows the quantification of the western blots of <FIG> and <FIG>. The responders could be easily identified in the western blots.

To determine whether a higher dose of onvansertib could reduce pTCTP in non-responders, blood cells of patients that were administered <NUM>/m<NUM> were analyzed. <FIG> shows western blots of the six patients described in <FIG> and <FIG>, treated at <NUM>/m<NUM> along with western blots of <NUM> non-responders that were treated at the higher dose. Even at a higher dose of onvansertib, the cells of non-responders did not show a decrease of pTCTP in response to treatment.

Levels of pTCTP in responders and non-responders were analyzed for differences pre- and post-treatment with onvansertib. As shown in <FIG>, there were no differences in pTCTP levels prior to treatment. Decrease in pTCTP at post-treatment was significantly higher in responders versus non-responders.

The methods described in Example <NUM> were used to determine the ability of onvansertib treatment, at increased dose, to inhibit phosphorylation of TCTP and the ability to use that inhibition to predict disease responsiveness to that treatment.

Blood samples were collected before, and <NUM> hours after, onvansertib treatment from patients enrolled in the trial described in Example <NUM>. The onvansertib treatments were <NUM>, <NUM>, <NUM>, or <NUM>/m<NUM>. pTCTP and TCTP were assessed by Western-Blot and % pTCTP (pTCTP/TCTP) was quantified.

In these studies, biomarker positivity was defined as ≥ <NUM>% decrease in % pTCTP from T=<NUM> to T=<NUM>.

Results are shown in <FIG>. The top and middle panels show examples of western blots of samples from patients with biomarker positive, and biomarker negative pTCTP inhibition, respectively. Nine out of the <NUM> evaluable patients (<NUM>%) were biomarker positive. Biomarker positivity was not dependent on onvansertib dose, pharmacokinetics, nor single-agent effects of LDAC or decitabine.

Of the biomarker positive patients, <NUM>-<NUM> and <NUM>-<NUM> were being treated with LDAC; <NUM>-<NUM>, <NUM>-<NUM>, and <NUM>-<NUM> were being treated with decitabine. Of the biomarker negative patients, <NUM>-<NUM> and <NUM>-<NUM> were treated with LDAC; <NUM>-<NUM>, <NUM>-<NUM> and <NUM>-<NUM> were treated with decitabine. This confirms that treatment with either LDAC or decitabine apparently does not affect the ability of a PLK1 inhibitor (here, onvansertib) to inhibit phosphorylation of a PLK1 target (here, TCTP).

Biomarker positive patients were being treated with onvansertib <NUM>, <NUM> or <NUM>/m<NUM> and biomarker negative patients were being treated with onvansertib <NUM>, <NUM>, <NUM> and <NUM>/m<NUM>. This indicates that biomarker positivity is not dependent on onvansertib dose. In addition, pharmacokinetics analysis showed no correlation between biomarker positivity and onvansertib concentration in blood.

The bottom panel of <FIG> shows the range of % pTCTP at post-dose relative to pre-dose for biomarker positive ("Target Engagement") and biomarker negative ("No Target Engagement") patients.

<FIG> show that biomarker positivity predicts response to treatment. <FIG> shows that biomarker positive patients had a significantly greater decrease in BM blasts compared to biomarker negative patients. As shown in <FIG>, six out of the nine biomarker positive patients had a decrease in BM blasts ≥ <NUM>%. Four patients had a complete response [CR] or CR with incomplete hematological recovery [CRi], as defined in Cheson et al. Among the <NUM> patients with objective responses (CR+CRi), <NUM> were biomarker positive and <NUM> had a <NUM>% decrease in pTCTP.

Claim 1:
A method of selecting cancer patients for treatment with onvansertib comprising
determining phosphorylation of serine <NUM> of translational control tumor protein (TCTP) in a cancer sample obtained from a patient, wherein the sample is a sample of cells of the cancer or a sample of cell-free TCTP in the serum of the patient
wherein phosphorylation is determined
(a) prior to exposure of the cells or cell-free TCTP to onvansertib; and
(b) after the cells or cell-free TCTP have been exposed to onvansertib;
and
selecting the patient for treatment with onvansertib if the phosphorylation is decreased after the cells or cell-free TCTP have been exposed to onvansertib compared to before they have been exposed to onvansertib;
wherein the cancer is acute myeloid leukemia (AML).