Compositions and Methods for Treating Cancer

Abstract

The small molecule compound IODVA1 has been found to have cytostatic activity against several transformed cell lines including Ras-driven cells. IODVA1 reduces cell-cell and cell-extracellular matrix interactions and reduces the growth of Ras-driven tumors. The applicant has also synthesized the compound NIRA2 and demonstrated in vitro and in vivo efficacy and potency against the Ph+(BCR-ABL1) B-ALL model and the colon adenocarcinoma xenograft model.

Description

[0001] Cross - Reference to Related Applications

[0002] This application hereby claims the benefit of the filing date of the same titled provisional patent application Ser. No. 62 / 842,839, filed May 3, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

[0003] Statement Regarding Federally Sponsored Research

[0004] This invention was made with government support under CA115611 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0005] In the United States and other developed countries, cancer is a serious public health problem. Currently, one in four deaths in the United States is due to cancer. However, many cancers do not respond to, or only minimally respond to, existing treatments, rendering current therapies ineffective. In addition, the emergence and recurrence of resistance mechanisms to current therapies remain a major clinical obstacle. Currently, the main therapies for cancer are surgery, radiotherapy, targeted and immunotherapy, and chemotherapy. Chemotherapeutic methods, such as anti-tumor antibiotics, alkylating agents, nitrosourea compounds, vinca alkaloids, steroid hormones, and antimetabolites, constitute most of the therapies available to oncologists. They have adverse side effects because they cannot distinguish between healthy and cancerous tissues. Despite progress in the field of cancer treatment, cancer remains a major health problem.

[0006] Accordingly, there is a pressing need in the art for compositions and methods for treating cancer. The present disclosure seeks to address this need in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] This application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee.

[0008] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the teachings of the present invention in any way.

[0009] Figure 1 . Identification of NSC124205. (A) Surface representation (left) of RasG60A in the GTP-bound form (PDB ID 1XCM), where the nucleotide is shown as red ball-and-stick and Mg 2+The ions are green spheres. The switch I region is magenta, while the switch II region is orange-red. Magnified surface used in the docking experiment (right). Water molecules are shown as red spheres. Figure prepared with the PyMol program. (B) NSC600805 and NSC124205 inhibit the growth of H292 and A549 cells. H292 and A549 lung adenocarcinoma cells were seeded at 10,000 cells per well in 24-well plates and treated in triplicate with 10 μM vehicle control or each of the 40 top-scoring NCI compounds. Cell proliferation was determined by MTS assay and plotted relative to the vehicle control at the 4-day time point. (C) Structures of compounds NSC600805 and NSC124205, as shown in PubChem, drawn using ChemDraw 18.0.

[0010] Figure 2 : Biochemical validation of NSC124205. (A) NSC124205 reduces acute AKT and ERK activation in NIH-3T3 cells. NIH-3T3 cells were starved for 24 hours, incubated with 15 μM DMSO vehicle control or the indicated NCI compound for 1 hour, stimulated with EGF for 5 minutes, and lysed with sample buffer. Lysates (30 μg) were resolved on 12% SDS-PAGE, transferred, and blotted for pAKT and pERK1 / 2. GAPDH was used as a loading control. (B) NSC124205 reduces the chronic levels of pAKT and pERK in Ras-activated cells. ST8814 cells were grown in RPMI medium 1640 supplemented with 10% FBS for 24 or 48 hours in the presence of the indicated concentration of NSC124205. Levels of pAKT and pERK were revealed by immunoblotting and quantified relative to the GAPDH level. (C) NSC124205 has no effect on ERK activation in cells expressing Ras G12V in NIH-3T3 cells. NIH-3T3 cells were stably transduced with full-length wild-type or G12V H-Ras. Cells were lysed in RIPA supplemented with protease and phosphatase inhibitors. Lysates (30 μg) were resolved on 12% SDS-PAGE, transferred, and blotted for HRas and GAPDH. 3T3 cells were serum-starved for 24 hours, incubated with 10 or 50 μM of the listed compounds for 2 hours, activated with EGF / serum for 5 minutes, lysed, normalized, separated by SDS-PAGE, and blotted for pERK. (D) Effect of NSC124205 on the colony-forming ability of NIH-3T3 cells expressing HRAS G12V in the presence of 5 and 25 μM of NSC124205. G12V3T3 cells were subjected to soft agar colony formation assay. Data represent the mean ± standard error of three experiments performed in quadruplicate. Statistical significance of differences between control and treated cultures was calculated by Student's t-test. NCI compounds 2 to 10 were from another branch of our drug discovery project.

[0011] Figure 3 .Chemical analysis of NSC124205. Chemical analysis of NSC124205. (A) NSC124205 is a mixture of compounds. Twenty microliters of a 1 mM NSC124205 solution was loaded onto a C18 column washed with buffer A (95% water, 5% acetonitrile, 0.1% formic acid), and a linear gradient was applied over 20 minutes using buffer B (95% acetonitrile, 5% water, 0.1% formic acid). Three peaks, 1a - 1c, eluted at 11.6 minutes, 12.8 minutes, and 13.4 minutes, respectively (upper panel). Under the same conditions, IODVA1 eluted as a single peak at 12.8 minutes. (B) Electrospray ionization (ESI) spectrum of IODVA1. (C) Proposed structure of IODVA1, where m / z is 370.1409 [M+H]+, and subsequently (Nishimura and Kitajima 1979) reported the reaction of guanidine derivatives with α-diketones. (D) 13 13C-NMR of IODVA1 synthesized in methanol-d4. Peaks corresponding to C's bearing hydroxyl groups are boxed. (E) IR spectrum of IODVA1. The expected stretches in the carbonyl region are boxed. (F) Electrospray ionization spectra of the three peaks at 11.6, 12.8, and 13.4 minutes, respectively. Structures of each component are shown. (G) MS-MS fragmentation of the 370.1409 peak of IODVA1. (H) 1 1H NMR of IODVA1 synthesized in methanol-d4. (I) Proposed mechanism for the reaction between 2-guanidino benzimidazole and α-pyrrolidone to generate structures A and B, NSC124205, and other products.

[0012] Figure 4.IODVA1 inhibits the proliferation of cancer model cells. (A) IODVA1 is an effective inhibitor of cell proliferation. MCF10A, MCF7, MDA-MB-231, and T47D cells were grown and counted in the presence of the indicated IODVA1 concentrations for 4 days or up to 7 days. Each point and bar are the mean ± stdev of 3 independent experiments, with 2 technical replicates in each experiment. (B) Colony numbers generated by MCF7, T47D, and MDA-MB-231 cells at the indicated IODVA1 concentrations. The results shown are the mean ± stdev of 2 independent experiments, with 4 technical replicates in each experiment. (C) IODVA1 inactivates Ras in the late stage of cultivation. Total Ras immunoprecipitated with GST-RafRBD from ST8814 cells treated with 0 or 2 μM IODVA1 at the indicated times showed that IODVA1 reduced Ras activation after 48 hours of treatment. A quantitative summary of 2 independent experiments is presented in the figure. n.s. – not significant, *-p<0.05, **-p<0.01, ****-p<0.0001. (D) IODVA1 inhibits the proliferation of ST8814 cells. ST8814 cells were grown in the presence of the indicated IODVA1 concentrations and counted daily for 4 days. Each point and bar are the mean and standard deviation of 3 independent experiments.

[0013] Figure 5: IODVA1-induced cytoskeletal changes. IODVA1 inhibits lamellipodia and circular dorsal ruffles (CDRs) formation and reduces Rac activation in MDA-MB-231 cells. (A) IODVA1 inhibits EGF-induced lamellipodia formation in MDA-MB-231 cells. MDA-MB-231 cells were plated on fibronectin-coated coverslips, serum-starved for 4 h, incubated with indicated concentrations of IODVA1 for 1 h, then stimulated with EGF (50 ng / mL), fixed, and stained with Phalloidin Alexa Fluor 594 (F-actin, pseudo-red) and DAPI (nuclei, pseudo-blue). Representative images show lamellipodia formation and enrichment of actin staining at the leading edge (white closed arrows) at 0 and 0.3 μM concentrations, while lamellipodia with lack of circular cell morphology at 1 and 3 μM. Note the evenly distributed phalloidin staining, where stress fibers are present in the cell bodies of 1 and 3 μM-treated cells (white open arrows), indicating quiescent cells. Scale bar = 10 μm. Results represent three independent experiments. (B) IODVA1 inhibits PDGF-induced CDR formation in 3T3 fibroblasts. NIH-3T3 cells were plated on fibronectin-coated coverslips, serum-starved for 4 h, incubated with indicated concentrations of IODVA1 for 1 h, then stimulated with PDGF (50 ng / mL), fixed, and stained with Phalloidin Alexa Fluor 594 (F-actin, pseudo-magenta) and DAPI (nuclei, pseudo-blue). Closed white arrows indicate circular dorsal ruffles and arrows indicate lack of the typical elongated morphology in stimulated fibroblasts. The percentage of cells with CDRs was counted as the number of cells with CDRs normalized to the total number of cells in the field. Approximately 150 cells were counted per experiment per condition. Cells with multiple CDRs were only counted once. Results represent three independent experiments. Scale bar = 10 μm. (C) MDA-MB-231 cells were incubated with indicated concentrations of IODVA1 (IO1) for 1 h, lysed, and incubated with GST-PAK-GBD (binds active Rac and Cdc42) and GST-Rhotekin RBD (binds active RhoA). Protein complexes were resolved on SDS-PAGE and immunoblotted with pan-Rac, Cdc42, or RhoA antibodies. The levels of active Rac (RacGTP, percentage relative to control), active Cdc42 (Cdc42GTP, percentage relative to control), and active RhoA (RhoAGTP, percentage relative to control) were quantified using ImageJ and ImageLab, and the combined data are shown as mean ± s.e.m. From at least 3 independent experiments.(D) Left panel – MDA-MB-231 cells were incubated with IODVA1 (IO1, 1 μM) for 30 min, lysed, and immunoblotted for pPAK1 (T423) / pPAK2 (T402). Lysates were loaded in duplicate. Shown results are mean ± s.e.m. of two independent experiments. Right panel – MDA-MB-231 cells were incubated with IODVA1 (IO1, 0.3 and 1 μM) for 30 min or 3 h, lysed, and immunoblotted for pPAK4 (S474) / pPAK5 (S602) / PAK6 (S560). Shown results are mean ± s.e.m. of two independent experiments. n.s. – not significant, * - p < 0.05, ** - p < 0.01, **** - p < 0.0001. (E) MDA-MB-231 cells were EGF-activated for 10 min, washed, treated with DMSO vehicle control or IODVA1 (3 μM) for 30 min, fixed, and stained for F-actin and nuclei (N = 3). Arrows point to lamellipodial structures. Images were taken at 100× magnification. Scale bar = 10 μm.

[0014] Figure 6: IODVA1 inhibits cell-matrix and cell-cell interactions. (A) IODVA1 blocks the spreading of MCF7 and MDA-MB-231 cells on fibronectin. MCF7, T47D, and MDA-MB-231 cells were seeded on fibronectin-coated coverslips for 10 minutes and then further incubated for 30 minutes in serum-free medium with the indicated concentrations of IODVA1, fixed, and visualized by bright-field microscopy. The area of individual cells was calculated from 6 random fields (total of at least 300 cells per treatment group). The results shown are the mean ± s.e.m. of a single experiment and represent three independent experiments. (B) Effect of IODVA1 treatment on spheroid formation in MCF10A, MCF7, T47D, and MDA-MB-231 cells. Left panel – Representative bright-field images of hanging-drop cultures of MCF10A, MCF7, T47D, and MDA-MB-231 cells grown in the absence (0 μΜ) and presence of IODVA1 (1 μΜ) before and after mechanical pipetting (trituration). Scale bar = 200 μm. Right panel, change in spheroid / aggregate size (indicated by spheroid diameter) due to IODVA1 treatment. The results shown are the mean ± stdev, N = 15. (C) IODVA1 treatment reduces proliferative capacity in an adhesion-free environment. MCF7, T47D, and MDA-MB-231 cells were grown in complete medium in the presence of IODVA1 or vehicle control in ultra-low attachment plates for 5 days. Aggregates and spheroids were dissociated with accutase and trituration, and the number of live cells was determined by trypan blue exclusion. The results shown are the combined mean ± stdev of two independent experiments. n.s. – not significant, * - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001.

[0015] Figure 7 : IODVA1 kinome inhibitory activity. The activity of 369 kinases was tested in duplicate in the presence of 0.5 μM IODVA1. Plotted is the remaining activity of replicate 1 versus 2 for each kinase, expressed as a percentage relative to the vehicle control set to 0%. Kinases with activity ratios that decreased or increased more than 3σ from the mean are indicated in red and green, respectively.

[0016] Figure 8 : IODVA1 inhibits tumor growth of human breast and lung cancer xenografts. (A) Orthotopic xenograft of MDA-MB-231 triple-negative breast cancer cells demonstrates that IODVA1 treatment reduces tumor growth. When the tumor volume reached 200 mm 3At 49 days post-injection, animals were initiated on vehicle (N = 6) or IODVA1 (N = 5) treatment and received three IODVA1 treatments per week for the next 28 days. (B) Tumors were also stained for Ki67 as a proliferation marker (top images). Compared to vehicle-treated tumors, IODVA1-treated tumors had a higher percentage of apoptotic cells as detected by cleaved caspase 3 immunofluorescence (CC3, bottom images). (C) Quantification of Ki67-positive cells and cleaved caspase 3-positive cells in the tumors shown in (B). (D) Xenograft tumors of H2122 lung cancer cells demonstrated that IODVA1 treatment reduced tumor growth. Animals were initiated on treatment when tumors were detectable at 10 days post-injection (*, p < 0.05). (E) H&E staining of representative H2122 tumors showed that IODVA1 decreased the number of mitotic cells in the tumors (top, arrows). Compared to animals treated with control vehicle, IODVA1-treated tumors also had fewer proliferating cells as determined by immunohistochemical staining for Ki67 (bottom), and increased intratumoral fibrosis. Representative images were taken at 100x magnification with a scale bar of 200 μm. (F) Quantification of Ki67+ cells in control and IODVA1-treated tumors shown in (E).

[0017] Figure 9 Repeated doses of IODVA1 did not cause toxicity in the hematopoietic system. Hemavet analysis of blood cell counts in peripheral blood collected from tumor-bearing breast cancer xenograft animals after 12 doses of IODVA1. No statistically significant changes in blood cell counts were detected between vehicle control and IODVA1-treated animals (N = 4, mean, SEM).

[0018] Figure 10 IODVA1 inhibits the proliferation and survival of BCR-ABL-expressing cells in vitro and in vivo and eradicates leukemic proliferating cells in secondary transplantation. (A) Human peripheral CD34 transduced with p190-BCR-ABL1 (gray lines, squares and black lines, inverted triangles) or Mieg3 empty vector (lilac lines, circles and triangles) virus +Hematopoietic cells were co-cultured on OP-9 stroma and incubated with vehicle or IODVA1 (IO1, 1 μM). Cell proliferation was evaluated by flow cytometry. (B) Cells were transduced and cultured as in (A), but incubated with vehicle or IODVA1 (IO1, 1 or 3 μM), and viability (%) was determined by trypan blue exclusion. (C) Kaplan–Meier plots showing the survival of p190-BCR-ABL1 leukemia mice after treatment with the indicated concentrations of vehicle control, IODVA1 (IO1), imatinib (IM), or combination in the pump. LDBM cells were transduced with a bicistronic p190-BCR-ABL1 / EGFP retrovirus and transplanted into recipient mice. After a preliminary assessment of the leukemia burden, drugs were delivered by subcutaneously implanted osmotic pumps for two-week periods. (D) Kaplan–Meier survival plots of secondary mouse transplants with 10 6 -fold cell dilutions. Bone marrow cells from mice treated with the indicated concentrations of vehicle, imatinib (IM), IODVA1 (IO1), or combination were transplanted into secondary recipients.

[0019] Figure 11: IODVA1 inhibits the proliferation and survival of BCR-ABL1-expressing cells in vitro and in vivo and eradicates leukemia proliferating cells in secondary transplantation. (A) Leukemic Ba / F3 cells transduced with p190-BCR-ABL1 (gray squares), p210-BCR-ABL1 (light gray triangles), or Mieg3 empty vector (black circles) were grown in the presence of 1 and 3 μM vehicle control or IODVA1 (IO1), and counted daily using trypan blue exclusion for 3 days. (B) IODVA1-dependent survival curves of Ba / F3 cells and Nalm-1 cells expressing empty vector (red circles) or p190-BCR-ABL1 (black circles). Data fitting was done in Prism version 8.4. (C) Ba / F3 cells expressing p190-BCR-ABL1 (gray line) or Mieg3 (black line) were grown for 1 day, treated with IODVA1 (IO1, 1 μM) for 1 day, then washed (black arrow) and cells were counted using trypan blue exclusion for 7 days. (D) Colony formation assay of Ba / F3 cells stably expressing p190-BCR-ABL1 in soft agar (0.25% noble agar, in RPMI / FBS / IL-3) in the presence of DMSO or IODVA1 (1 or 10 μM). Colonies were allowed to form for 10 days and then stained with iodonitro tetrazolium (1 mg / mL). Data represent at least three independent experiments in triplicate. Note the smaller colony size in the 1 μM IODVA1 treatment group. (E) Analysis of the leukemia burden (%) of pre-treatment (upper left panel) and treated mice at the indicated treatment times by flow cytometry of bone marrow aspirates as a population containing B220 + / CD43 + pro-B cells. (F) Figure 11 Count of residual leukemia (EGFP + -BCR-ABL1) cells in the peripheral blood of secondary transplantation mice in D at weeks 3 and 5. (G) Kaplan-Meier survival plot of secondary mouse transplantation with 0.3 x 10 6 cell dilutions. (H) Count of residual leukemia (EGFP + -BCR-ABL1) cells in the peripheral blood of secondary transplantation mice in (G) at weeks 3 and 5. (I&J) Similar to G&H, but with 0.1 x 10 6 cell dilutions.

[0020] Figure 12 : IODVA1 but not imatinib improves the survival rate in a TKI-resistant B-ALL mouse model. LDBM cells were transduced with TKI-resistant p210-BCR-ABL1 (T315I) (gatekeeper mutant) and transplanted into recipient mice asFigure 10 As shown. The pump was introduced into mice by surgery (N = 5 for each treatment group), and the treatment continued for 28 days or two rounds of the pump. After 28 days, the mice were monitored without any additional treatment. (A) Kaplan-Meier survival plot of imatinib-resistant mice. The pumps carried vehicle control (black line), 0.5 mM imatinib (IM, gray line), or 0.5 mM IODVA1 (IO1, lavender line). (B) Flow cytometry analysis of leukemia progenitor (EGFP+) B cells in peripheral blood (PB) at the indicated weeks. At week 5, week 7, and week 10, only the mice treated with IODVA1 remained alive for analysis. (C) Pharmacodynamic evaluation of leukemia progenitors (%) from mice treated with vehicle control (black), imatinib (gray), or IODVA1 (lavender) for 2 weeks using phospho-flow analysis with the indicated Rac-dependent and -independent effectors. *p ≤ 0.05, **p ≤ 0.01.

[0021] Figure 13 : IODVA1 reduces Rac activation and signaling. (A) As shown, Ba / F3 cells expressing p190-BCR-ABL1 were treated with IODVA1 (3 μM), and the level of active Rac (Rac-GTP) was evaluated by pull-down using GST-PAK-GBD, followed by immunoblotting (upper panel) and densitometric quantification (lower panel). (B) Flow cytometry analysis of pJNK, pS6, p4EBP, pPAK1, and pAKT in Ba / F3 cells expressing Mieg3 empty vector (light blue and dark blue) or p190-BCR-ABL1 (light orange and dark orange) and treated with vehicle control or IODVA1 (3 μM) for 30 minutes. (C) Representative histogram data of cell cycle analysis of Ba / F3 cells expressing p190-BCR-ABL and treated with vehicle control or IODVA1 (1, 3, and 10 μM) for 20 hours. (D) Quantification of the average number of colonies of bone marrow wild-type (black) and Rac1Δ / Δ+Rac2 - / - (red) p190-BCR-ABL1 leukemia cells. ns – not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (E) As Figure 13As shown in A, cells were cultured for a fixed time (1 hour) at the specified IODVA1 concentration. (F) Ba / F3 cells expressing p190-BCR-ABL1 were treated with IODVA1 (3 μM) and lysed at the specified times. Cell lysates were separated by SDS-PAGE and immunoblotted for pPAK (T423), pBAD (S136), and BAD. (G) Ba / F3 cells expressing p190-BCR-ABL1 were treated with IODVA1 (3 μM), and the levels of active Cdc42 (Cdc42-GTP) and Rho (Rho-GTP) were evaluated by pull-down using GST-PAK-GBD and GST-Rhotekin, respectively, at the specified times, followed by immunoblotting (left panel) and densitometry quantification (right panel). (H) GFP + Morphology of leukemia colonies (left panel). Rac1Δ / Δ+Rac2 - / - Protein blot analysis of Rac1 and Rac2 protein expression in cells (right panel). (I) Intrinsic (blue line) and p50GAP-stimulated GTP hydrolysis reactions in the presence (red line) or absence (black line) of IODVA1. (J) Precipitation assay of liposomal Rac1-GDP in the presence of IODVA1 (2 μM). Rac1 was visualized by immunoblotting from the pellet (p) and soluble (s) fractions. (K) Stopped-flow measurements of the interaction of GDI (10 μM) with fluorescently labeled Rac1 in the absence (black line) or presence (orange line) of IODVA1.

[0022] Figure 14 : IODVA1 targets Vav3 in vitro and in vivo. (A) Ba / F3 cells expressing the empty vector Mieg3 or p190-BCR-ABL1 were treated with vehicle control or IODVA1 (IO1, 3 μM) for 30 minutes and incubated with GST-Rac and glutathione beads. The beads were washed and the protein complexes were separated by SDS-PAGE and immunoblotted for pVav3. Input Vav3 was used as a control. (B) Binding affinity (K d ) between IODVA1 and Vav3 (green), LARG (brown), and RacGDP (blue). Microscale thermophoresis signals expressed as fractional occupancy were plotted against IODVA1 (0.1 nM–20 μM) and fitted to generate K d . Error bars = SD; N = 3. (C) Bone marrow wild-type (black) and Vav3 - / -(Lavender) Quantification of the average number of colonies produced by p190 - BCR - ABL1 leukemia cells (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns, not significant). (D) Wild - type (black bars and gray bars) and Vav3 expressing p190 - BCR - ABL and treated with vehicle control or IODVA1 (IO1, 3 μM) for 20 hours - / - (Dark purple and light purple) Representative histogram data of cell cycle analysis of bone marrow cells. (E) Quantification by densitometry of the pVav3 band in A. (F) Shows wild - type or Vav3 Figure 14 - / - Kaplan - Meier plot of the survival of p190 - BCR - ABL1 leukemia mice after treatment with osmotic pumps implanted subcutaneously and carrying vehicle control or IODVA1 (IO1, 1 mM). (G) Counts of residual leukemia (EGFP + - BCR - ABL1) cells in the mice from (F) at 1 week and 2 weeks after treatment (percentage of leukemia progenitors in peripheral blood). (H) Pharmacodynamic evaluation of leukemia progenitors (%) from wild - type or Vav3 - deficient mice with p190 - BCR - ABL1 leukemia treated with vehicle control (dark gray and light gray) or IODVA1 (IO1, dark purple and light purple) after 2 weeks of treatment using phospho - flow analysis of the indicated effectors (*p ≤ 0.05; **p ≤ 0.01).

[0023] Figure 15 : Expression of transgenic Vav3 renders Vav3 - deficient cells sensitive to IODVA1 again. (A) Vav3 domain structure, calmodulin homology (CH), acidic region (Ac), Dbl homology (DH), Pleckstrin homology (PH), zinc finger (ZF), Src homology 2 and 3 (SH2 / SH3). (B) Wild - type (Vav3 + / + ) and Vav3 - deficient (Vav3 - / - ) p190 - BCR - ABL1 leukemia bone marrow cells expressing empty vector, full - length Vav3 or ΔCH mutant and treated with 5 (red) or 10 μM (dark gray) vehicle control (black) or IODVA1 for 18 hours, representative histogram data of cell cycle analysis. (C) Wild - type bone marrow and Vav3 - / - Quantification of the average number of colonies produced by p190 - BCR - ABL1 leukemia bone marrow cells expressing empty vector, full - length Vav3 or ΔCH mutant and treated with 1 (light orange), 5 (red) and 10 μM (dark gray) vehicle control (black) or IODVA1. ***p ≤ 0.001.

[0024] Figure 16 ​: IODVA1 reduces the level of pVav3 in in vitro and in vivo models of triple-negative breast cancer. (A) Immunoblotting and quantification of Vav3 protein in lysates of MDA-MB-231 triple-negative breast cancer cells stably expressing scrambled or Vav3-targeting shRNA. Viability of MDA-MB-231 cells stably expressing shVav3 in the presence of IODVA1 (0 - 1 μM). Cells were grown in the presence of IODVA1 and counted by trypan blue exclusion at the indicated time points. (B) MDA-MB-231 cells were incubated with IODVA1 (3 μM) for 15 minutes, and the level of phosphorylated Vav3 (pY173) was evaluated by immunoblotting. (C) Immunohistochemical staining of phosphorylated Vav3 in tissues from MDA-MB-231 xenografts treated with vehicle control or IODVA1.

[0025] Figure 17 : IODVA1 reduces the viability of cells from pediatric patients with relapsed and de novo Ph + leukemia. Patient-derived xenograft (PDX) cells were co-cultured ex vivo on MS-5 or OP-9 stromal cells and treated with dasatinib (Das, ABL1 inhibitor), ruxolitinib (Rux, JAK inhibitor), a combination of dasatinib and ruxolitinib (Das + Rux), abemaciclib (CDK inhibitor), or IODVA1, and viability was evaluated. (A) Representative viability and colony-forming ability of cells from patient #2018-136 treated with IODVA1 (IO1). (B) Viability of cells from patient #2017-58 treated with the indicated agents and (C) viability of cells from patient #2017-129 with a BCR-ABL1 (T315I) mutation. Note that IODVA1 lacks toxicity to normal stromal cells (black arrows) in the figure.

[0026] Figure 18 : IODVA1 reduces the viability of leukemia cells from relapsed and de novo Ph-like and MLL pediatric patients. Patient-derived xenograft (PDX) cells were co-cultured ex vivo on MS-5 or OP-9 stromal cells and treated with dasatinib (Das, ABL1 inhibitor), ruxolitinib (Rux, JAK inhibitor), a combination of dasatinib and ruxolitinib (Das + Rux), abemaciclib (CDK inhibitor), or IODVA1, and viability was evaluated. (A) to (E) De novo Ph-like leukemia cells. (F) Leukemia cells from MLL / AF9 and relapsed MLL / AF1q patients (G - H).

[0027] Figure 19: NIRA2 inhibits the proliferation and survival of cells expressing BCR-ABL1 in vitro. (A) Leukemic Ba / F3 cells transduced with p190-BCR-ABL1 (red circles) or Mieg3 empty vector (black circles) were grown in the presence of the indicated concentrations of vehicle control or NIRA2 and counted daily using trypan blue exclusion for 3 days. (B) Plotting the NIRA2-dependent survival rate of Ba / F3 cells expressing p190-BCR-ABL1 from (A) at the 24-hour time point yielded an EC50 of 42.2 nM. Data fitting was done in Prism version 8.4.

[0028] Figure 20 : NIRA2 kinase panel inhibitory activity. The activities of 485 kinases (ThermoFisher) were tested in duplicate in the presence of 0.5 μM NIRA2. Plotted is the remaining activity of replicate 1 versus replicate 2 for each kinase, expressed as a percentage relative to the vehicle control set to 0%. Kinases with an activity ratio decreased or increased by more than 3σ from the mean are indicated.

[0029] Figure 21 : NIRA2 inhibits tumor growth of murine colon adenocarcinoma xenografts. Orthotopic xenotransplantation of MC38 colon adenocarcinoma cells demonstrated that NIRA2 treatment reduced tumor growth. When the tumor volume reached 100 - 200 mm 3 (on day 12 after injection), the animals were started on treatment with vehicle, IODVA1, or NIRA2 (N = 10 per group) and received 5 treatments per week for two weeks. Representative pictures of the spleen and excised tumors are shown. Detailed Description

[0030] Definitions

[0031] Unless otherwise indicated, terms shall be understood according to the ordinary usage of those of ordinary skill in the relevant art. In case of conflict, the present document (including definitions) shall prevail. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used to practice or test the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0032] As used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods and reference to "a dose" includes reference to one or more doses known to those of skill in the art and their equivalents, etc.

[0033] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by a person of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measuring system. For example, in accordance with the practice in the art, "about" can mean within 1 or more standard deviations. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude of the value, preferably within 5-fold and more preferably within 2-fold. When a particular value is described in this application and in the claims, unless otherwise indicated, the term "about" should be assumed to mean that the particular value is within an acceptable error range.

[0034] As used herein, the term "effective amount" means an amount of one or more active ingredients sufficient to exhibit a desired effect. This includes therapeutic and prophylactic effects. When applied to an individual active ingredient administered alone, the term refers to that individual active ingredient. When applied to a combination, the term refers to the combined amount of the active ingredients that results in a therapeutic effect, whether administered continuously in combination or simultaneously.

[0035] The terms "individual", "host", "subject" and "patient" are used interchangeably to refer to an animal that is the subject of treatment, observation and / or experimentation. Generally, the term refers to a human patient, but the methods and compositions can equally apply to non-human subjects, such as other mammals. In some embodiments, the term refers to a human. In some embodiments, the term can refer to a child.

[0036] The term "pharmaceutically acceptable salt" or "its pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic acid or base. Suitable pharmaceutically acceptable salts include metal salts such as aluminum salts, zinc salts, alkali metal salts such as lithium salts, sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts; organic salts; salts of free acids and bases; inorganic salts such as sulfates, hydrochlorides and hydrobromides; and other salts that are currently widely used in pharmaceutical applications and are listed in sources well-known to those skilled in the art (such as The Merck Index). Any suitable component can be selected to prepare the salts of the active drugs discussed herein, provided that the composition is non-toxic and does not substantially interfere with the desired activity. In addition to salts, pharmaceutically acceptable precursors and derivatives of the compounds can also be employed. Pharmaceutically acceptable amides, lower alkyl esters and protected derivatives of the disclosed active substances can also be suitable for the compositions and methods disclosed herein. The salts of the compounds of the present disclosure can be formed between an acid and a basic group of the compound (such as an amino functional group) or between a base and an acidic group of the compound (such as a carboxyl functional group). According to another embodiment, the compound is a pharmaceutically acceptable acid addition salt. Acids commonly used to form pharmaceutically acceptable salts include inorganic acids such as hydrogenbisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, and organic acids such as p-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, and related inorganic and organic acids.Accordingly, such pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, caprates, heptanoates, propiolates, oxalates, malonates, succinates, octanedioates, decanedioates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, terephthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, β-hydroxybutyrates, glycolates, maleates, tartrates, mesylates, propionates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates and other salts. In one embodiment, the pharmaceutically acceptable acid addition salts include acid addition salts formed with inorganic acids such as hydrochloric acid and hydrobromic acid, and in particular acid addition salts formed with organic acids such as maleic acid.

[0037] As used herein, the term "treat", "treating" or "treatment" refers to alleviating, reducing or ameliorating the symptoms of a disease or disorder, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of the symptoms, inhibiting the disease or disorder, arresting the development of the disease or disorder, alleviating the disease or disorder, causing regression of the disease or disorder, alleviating the conditions caused by the disease or disorder, or methods for prophylactically and / or therapeutically halting the symptoms of the disease or disorder.

[0038] The term "carrier" as applied to the pharmaceutical compositions of the present disclosure refers to a diluent, excipient or vehicle with which the active compound is administered. Such pharmaceutical carriers can be sterile liquids such as water, saline solutions, aqueous glucose solutions, aqueous glycerol solutions and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" (any edition).

[0039] As used herein, the term "compound" is also intended to include any salts, solvates or hydrates thereof.

[0040] The term "alkyl" includes straight-chain, branched-chain or cyclic alkyls, such as but not limited to methyl, ethyl, propyl, butyl, trifluoromethyl and tetradecyl.

[0041] The term "alkoxy" includes straight-chain, branched-chain or cyclic alkoxys, such as but not limited to methoxy, ethoxy, propoxy, butoxy, 2-methoxyethoxy, sec-butoxy, hexyloxy and 2-ethylhexyloxy, tetradecyloxy.

[0042] The term "aryl" encompasses monocyclic and polycyclic aryls containing only carbon in the first ring. The term "monocyclic aryl" refers to phenyl (where the ring contains only carbon), and the term "polycyclic aryl" refers to naphthyl and anthracenyl, a benzene ring fused with at least a second ring, and a naphthalene ring fused with at least a third ring. In the case of a polycyclic aryl composed of a benzene ring fused with a second or third ring or a naphthalene ring fused with a third ring, the additional ring can be an aromatic or non-aromatic carbocyclic or heterocyclic ring, provided that in such a case, the point of attachment will be connected to the carbocyclic aromatic ring. For example, a subset of said aryls is a polycyclic aryl where the second ring is a "heteroaryl" containing carbon atoms and at least one heteroatom selected from the group consisting of O, N, and S (provided that O and S cannot be adjacent in the same ring). Alternatively, the ring carbon atoms of the second and / or third additional rings can be replaced by a carbonyl [-C(=O) group] (e.g., when such rings are non-aromatic). "Substituted aryl" refers to an aryl substituted at any point of attachment of any ring by one or more substituents, preferably 1 to 4 substituents (more preferably 1 or 2 substituents), said substituents being selected from alkyl, substituted alkyl, and the substituents described above for substituted alkyl.

[0043] Thus, examples of aryls of interest for forming the compounds of the present invention include:

[0044]

[0045] And, additionally, similar structures.

[0046] The terms “heterocycle,” “heterocyclic,” and “heterocyclo” refer to fully saturated, partially unsaturated, or fully unsaturated cyclic groups that contain an aromatic (i.e., “heteroaryl”) moiety (e.g., a 3- to 7-membered monocyclic, 7- to 11-membered bicyclic, or 10- to 16-membered tricyclic system) and that have at least one heteroatom in at least one carbon-containing ring. Thus, the term “heteroaryl” is a subset of heterocyclic groups. Each ring of a heterocyclic group containing a heteroatom may have 1, 2, 3, or 4 heteroatoms selected from nitrogen, oxygen, and / or sulfur atoms, where the nitrogen and sulfur heteroatoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized, provided that sulfur and oxygen are not adjacent to each other in the ring. (The term “heteroaryl” refers to a heteroaryl bearing a quaternary nitrogen atom and thus a positive charge.) Further, one or more (preferably one) carbon ring atoms of the heterocycle may be replaced by a carbonyl group (i.e., —C(═O)—) if valency permits. A heterocyclic group may be attached to the remainder of the molecule at any heteroatom or carbon atom of the ring or ring system.

[0047] Exemplary monocyclic heterocyclic groups include groups selected from the group consisting of oxirane, azetidinyl, pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazetidinyl, azetidinyl, hexahydrodiazepinyl, 4-piperidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazolyl, tetrazolyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolanyl, and tetrahydro-1,1-dioxothienyl, among others.

[0048] Exemplary bicyclic heterocyclic groups include groups selected from the group consisting of: indolyl, isoindolyl, benzothiazolyl, benzodioxazolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, quinuclidinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, benzofurazanyl, chromonyl, coumarinyl, benzopyranyl, cinnamyl, quinoxalinyl, indazolyl, pyrrolopyridinyl, furanopyridinyl (such as furan[2,3-c]pyridinyl, furan[3,2-b]pyridinyl] or furan[2,3-b]pyridinyl), dihydrobenzodioxinyl, dihydrobenzothiophenedioxideyl, dihydroisoindolyl, dihydroindolyl, dihydroquinolinyl, dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl), triazinylazepinyl, tetrahydroquinolinyl, etc. Exemplary tricyclic heterocyclic groups include carbazolyl, benzyl, phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyl, etc.

[0049] The term "heterocyclene" refers to a divalent heterocyclic group as defined above.

[0050] "Substituted heterocycle, substituted heterocyclic, and substituted heterocyclo" (such as "substituted heteroaryl") refers to a heterocycle (heterocycle, heterocyclic, or heterocyclo) group substituted at any available attachment point with one or more substituents (preferably 1 to 4 substituents), where the substituents are selected from the substituents described above for substituted cycloalkyl.

[0051] The term "group" is intended to cover not only the unsubstituted form of the substituent but also its form further substituted with any one or more of the substituents mentioned herein, provided that the substituent does not destroy the properties required for practical use. Suitably, the substituent can be a halogen or can be attached to the rest of the molecule through a carbon, nitrogen, oxygen, or sulfur atom.

[0052] In one aspect, the compositions that can be used according to the disclosed methods can include a compound having the following structure:

[0053] (referred to herein as "Compound 1" or "IODVA1 compound") and a pharmaceutically acceptable carrier;

[0054] where A = NH, NR8, S, O, C = C, N = C, C = N

[0055] where R1 and R2 are independently a substituted or unsubstituted aryl or heteroaryl ring

[0056] wherein R3 = single-substituted or multi-substituted with H, D, halogenated, CN, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylamino or C1-C4 mercapto

[0057] wherein R8 = H, M;

[0058] and all of its tautomers.

[0059] In one aspect, the compound can have the following structure

[0060]

[0061] wherein A = NH, S

[0062] wherein R1, R2 are independently a substituted or unsubstituted phenyl, pyridyl, furyl, pyrimidinyl, triazinyl or diazinyl ring

[0063] wherein R3 = single-substituted or multi-substituted with H, D, halogenated, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et or Pr;

[0064] and all of its tautomers.

[0065] In one aspect, the compound can have the following structure

[0066]

[0067] and wherein R1, R2 are independently a substituted or unsubstituted phenyl, pyridyl, furyl, pyrimidinyl ring

[0068] wherein R3 = single-substituted or multi-substituted with H, D, halogenated, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et or Pr,

[0069] and all of its tautomers.

[0070] In one aspect, the compound can have the following structure

[0071]

[0072] wherein R4-R17 are independently selected from H, D, halogenated, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et or Pr,

[0073] and all of its tautomers.

[0074] In one aspect, the compound can have the following structure

[0075]

[0076] wherein R4 - R17 are independently selected from H, D, halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et or Pr,

[0077] and all of their tautomers.

[0078] In one aspect, the compound may have the following structure

[0079]

[0080] wherein R4 - R7 are independently selected from H, D, F, Cl, CN, OH, OMe, SMe, Me or Et;

[0081] wherein R9 - R17 are independently selected from H, D, F, Cl, CN, OH, OMe, SO2Me, NHMe, NMe2, Me or Et;

[0082] and all of their tautomers.

[0083] In one aspect, the compound may have the following structure

[0084]

[0085] wherein R4 - R7 are independently selected from H, D, F, Cl, CN, OH, OMe, SMe, Me or Et;

[0086] wherein R9 - R17 are independently selected from H, D, F, Cl, CN, OH, OMe, SO2Me, NHMe, NMe2, Me or Et,

[0087] and all of their tautomers.

[0088] In one aspect, the compound may have the following structure

[0089]

[0090] wherein R4 - R7 are independently selected from H, D, F, OH, OMe, Me;

[0091] wherein R9 - R17 are independently selected from H, D, F, Cl, CN, OH, OMe, SO2Me, NHMe, NMe2, Me or Et,

[0092] and all of their tautomers.

[0093] In one aspect, the compound may have the following structure

[0094]

[0095] wherein R5 and R6 are independently selected from H, D, F, Cl, OH, OMe or Me;

[0096] wherein R9 - R17 are independently selected from H, D, F, Cl, OH, OMe or Me;

[0097] wherein each ring bears ≤ 2 non - H substituents;

[0098] and all of its tautomers.

[0099] In one aspect, the compound can have the following structure

[0100] and all of its tautomers.

[0101] In one aspect, the compound can have the following structure

[0102] and all of its tautomers.

[0103] In one aspect, a composition is disclosed, which comprises a compound having the following structure

[0104] and a pharmaceutically acceptable carrier.

[0105] In one aspect, the compound can have the following structure

[0106]

[0107] wherein A is selected from NH, NR8, S, O, C═C, N═C, C═N; wherein R1 and R2 are independently a substituted or unsubstituted *aryl or heteroaryl ring; wherein R3 is mono - substituted or poly - substituted with H, D, halo, CN, C1 - C4 alkyl, C1 - C4 alkoxy, C1 - C4 alkylsulfonyl, C1 - C4 alkylamino or C1 - C4 mercapto; wherein R8 is H or Me; and all of its tautomers.

[0108] In one aspect, the compound can have the following structure

[0109]

[0110] wherein A is NH or S; wherein R1 and R2 are independently a substituted or unsubstituted phenyl, pyridyl, furyl, pyrimidinyl, triazinyl or diazinyl ring; wherein R3 is mono - substituted or poly - substituted with H, D, halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et or Pr; and all of its tautomers.

[0111] In one aspect, the compound can have the following structure

[0112]

[0113] wherein R1 and R2 are independently a substituted or unsubstituted phenyl, pyridyl, furyl, pyrimidinyl ring; wherein R3 is mono- or polysubstituted with H, D, halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et or Pr; and all of its tautomers.

[0114] In one aspect, the compound can have the following structure

[0115]

[0116] wherein R4 - R17 are each independently selected from H, D, halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et or Pr; and all of its tautomers.

[0117] In one aspect, the compound can have the following structure

[0118]

[0119] wherein R4 - R17 are each independently selected from H, D, halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et or P; and all of its tautomers.

[0120] In one aspect, the compound can have the following structure

[0121]

[0122] wherein R4 - R7 are each independently selected from H, D, F, Cl, CN, OH, OMe, SMe, Me or Et; wherein R9 - R17 are each independently selected from H, D, F, Cl, CN, OH, OMe, SO2Me, NHMe, NMe2, Me or Et; and all of its tautomers.

[0123] In one aspect, the compound can have the following structure

[0124]

[0125] wherein R4-R7 = H, D, F, Cl, CN, OH, OMe, SMe, Me or Et; wherein R9-R17 = H, D, F, Cl, CN, OH, OMe, SO2Me, NHMe, NMe2, Me or Et, and all of their tautomers.

[0126] In one aspect, the compound may have the following structure

[0127]

[0128] wherein R4-R7 are independently = H, D, F, OH, OMe, Me; wherein R9-R17 may independently be selected from H, D, F, Cl, CN, OH, OMe, SO2Me, NHMe, NMe2, Me or Et, and all of their tautomers.

[0129] In one aspect, the compound may have the following structure

[0130]

[0131] wherein R4-R7 are independently = H, D, F, Cl, OH, OMe or Me; wherein R9-R12 and R14-R17 may independently be selected from H, D, F, Cl, OH, OMe or Me. Each ring bears ≤ 2 non-H substituents; and all of their tautomers.

[0132] In one aspect, a composition is disclosed, which comprises

[0133] and all of their tautomers, and a pharmaceutically acceptable carrier.

[0134] In one aspect, a composition is disclosed, which comprises

[0135] and all of their tautomers, and a pharmaceutically acceptable carrier.

[0136] In one aspect, a composition is disclosed, which comprises a compound referred to herein as a NIRA2 class compound. The composition may comprise a compound having the following structure:

[0137]

[0138] wherein R1, R2 and R4 are independently selected from H, D, halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et or Pr, and all of their tautomers, wherein R3 is mono- or polysubstituted with H, D, halo, CN, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylsulfonyl; C1-C4 alkylamino, or C1-C4 mercapto; wherein A is selected from NH, NR8, S, O, C═C, N═C, C═N, wherein R8 is H or Me, wherein each Q is independently selected from N, C and S; and a pharmaceutically acceptable carrier.

[0139] In one aspect, the compound may have the following structure

[0140]

[0141] wherein R5-R20 are independently selected from H, D, halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NMe2, Me, Et or Pr, wherein each Q is independently selected from N, C and S, and all of their tautomers; and a pharmaceutically acceptable carrier.

[0142] In one aspect, the compound may have the following structure

[0143]

[0144] wherein R1, R2 and R4 are independently selected from H, D, halo, CN, OH, OMe, OEt, SMe, SEt, SO2Me, NHMe, NMe2, Me, Et or Pr, and all of their tautomers; wherein R3 is mono- or polysubstituted with H, D, halo, CN, C1-C4 alkyl; C1-C4 alkoxy; C1-C4 alkylsulfonyl; C1-C4 alkylamino; or C1-C4 mercapto; wherein A is selected from NH, NR8, S, O, C═C, N═C, C═N, wherein R8 is H or Me, wherein each Q is independently selected from N, C and S; and a pharmaceutically acceptable carrier.

[0145] In one aspect, the compound may have the following structure

[0146] (“NIRA2”) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In one aspect, the compound may have the following structure

[0147] or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0148] In one aspect, any one of the above compositions may be free or substantially free of one or both of the following compounds:

[0149]

[0150] In one aspect, a composition is disclosed that comprises

[0151] (NSC600805) and a pharmaceutically acceptable carrier.

[0152] In one aspect, a method of treating cancer in an individual in need thereof is disclosed. The method can comprise the step of administering to the individual a compound or composition disclosed herein.

[0153] In one aspect, the cancer can be a solid tumor. The treating step can effect a reduction in tumor volume.

[0154] In one aspect, the treating step can effect a reduction in cancer cell proliferation. In one aspect, the treating step can effect an increase in cancer cell death.

[0155] In one aspect, the cancer can be selected from leukemia, preferably ALL, AML or MLL, chemotherapy-resistant leukemia, immunotherapy-resistant leukemia, relapsed leukemia and other targeted-therapy-resistant leukemia. In one aspect, the cancer can be selected from adenocarcinoma, breast cancer. In one aspect, the cancer can be a cancer in which Vav3 is overexpressed, such as prostate cancer, ovarian cancer, endometrial cancer, thyroid cancer, lung cancer (specifically, non-small cell lung cancer), colorectal cancer, pancreatic cancer and cervical cancer. In one aspect, the cancer can be a Ras-driven cancer, including RASopathy, e.g., NF1 or MPNST. In one aspect, the cancer is any cancer that overexpresses Vav3.

[0156] In another aspect, a method of treating cancer based on the status of Vav3 expression is disclosed. In this aspect, the method can comprise the steps of: determining the Vav3 level in a biopsy obtained from an individual's cancer; and administering to the individual a composition as described herein, wherein the Vav3 level is elevated compared to a control.

[0157] Pharmaceutical composition

[0158] The composition can be administered in oral dosage forms such as tablets, capsules (each containing a sustained release or timed release formulation), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The composition can also be administered intravenously (bolus or infusion), intraperitoneally, subcutaneously, intralesionally, or intramuscularly, all in dosage forms well known to those of ordinary skill in the pharmaceutical art. The composition can be administered by the intranasal route using a suitable intranasal vehicle, or by the transdermal route, for example, using a conventional transdermal skin patch. The dosing regimen using a transdermal delivery system can be continuous rather than intermittent throughout the dosing regimen.

[0159] In one aspect, the pharmaceutical composition is isotonic with the recipient's blood or other body fluids. Sodium tartrate, propylene glycol, or other inorganic or organic solutes can be used to obtain the isotonicity of the composition. Examples include sodium chloride. Buffering agents such as acetic acid and salts, citric acid and salts, boric acid and salts, and phosphoric acid and salts can be employed. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (such as Ringer's dextrose-based supplements), and the like.

[0160] The viscosity of the pharmaceutical composition can be maintained at a selected level using pharmaceutically acceptable thickening agents. Methylcellulose is useful because it is readily available, economical, and easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. In some embodiments, the concentration of the thickening agent will depend on the thickening agent selected. An amount that will achieve the selected viscosity can be used. Viscous compositions are generally prepared from solutions by adding such thickening agents.

[0161] Pharmaceutically acceptable preservatives can be employed to increase the shelf life of the pharmaceutical composition. Benzyl alcohol may be suitable, but a variety of preservatives can also be used, including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride. The suitable concentration of the preservative is generally from about 0.02% to about 2% based on the total weight of the composition, but greater or lesser amounts may be required depending on the reagent selected. As described above, reducing agents can be advantageously used to maintain the good shelf life of the formulation.

[0162] On the one hand, the active agents provided herein can be mixed with suitable carriers, diluents, or excipients (such as sterile water, physiological saline, glucose, etc.), and can contain auxiliary substances, such as wetting agents or emulsifiers, pH buffers, gelling or viscosity enhancing additives, preservatives, flavoring agents, coloring agents, etc., depending on the route of administration and the desired formulation. See, for example, "Remington: The Science and Practice of Pharmacy", Lippincott Williams & Wilkins; 20th Edition (June 1, 2003) and "Remington's Pharmaceutical Sciences", Mack Pub. Co.; 18th and 19th Editions (December 1985 and June 1990, respectively). Such formulations can contain complexing agents, metal ions, polymers (such as polyacetic acid, polyglycolic acid, hydrogels, dextrans, etc.), liposomes, microemulsions, micelles, monolayers or multilayers of vesicles, red blood cell ghosts, or spheroblasts. Suitable lipids for liposomal formulations include, but are not limited to, monoglycerides, diglycerides, thioesters, lysolecithin, phospholipids, saponins, bile acids, etc. The presence of such additional components may affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate, and thus are selected according to the intended application so that the properties of the carrier are suitable for the selected route of administration.

[0163] For oral administration, the pharmaceutical composition can be provided as tablets, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft gelatin capsules, syrups or elixirs. The compositions for oral use can be prepared by any method known in the art for manufacturing pharmaceutical compositions and can contain one or more of the following agents: sweetening agents, flavoring agents, coloring agents, and preservatives. The aqueous suspensions can contain the active ingredient mixed with excipients suitable for the preparation of aqueous suspensions.

[0164] The formulations for oral use can also be provided as hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent (such as calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules. In the soft gelatin capsules, the active agent can be dissolved or suspended in a suitable liquid, such as water or an oil medium, such as peanut oil, olive oil, fatty oils, liquid paraffin, or liquid polyethylene glycol. Stabilizers and microspheres formulated for oral administration can also be used. The capsules can include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer (such as glycerol or sorbitol). The push-fit capsules can contain the active ingredient, which is mixed with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer.

[0165] The tablets can be uncoated or can be coated by known methods to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a delayed release material such as glyceryl monostearate can be used. When administered in solid form (such as in tablet form), the solid form generally comprises from about 0.001 wt.% or less to about 50 wt.% or more of the active ingredient, for example, about 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 wt.% to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or 45 wt.%.

[0166] The tablets can contain the active ingredient admixed with non-toxic pharmaceutically acceptable excipients including inert materials. For example, the tablets can be prepared by compressing or molding, optionally with one or more additional ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in free-flowing form (such as a powder or granules), which is optionally mixed with a binder, lubricant, inert diluent, surfactant or dispersant. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0167] In some embodiments, each tablet or capsule contains from about 1 mg or less to about 1,000 mg or more of the active agent provided herein, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg to about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 or 900 mg. In some embodiments, the tablets or capsules are provided in a dosage range to allow for fractional dosing. Thus, the dose and the number of doses administered per day can be conveniently selected to suit the patient. In certain embodiments, two or more therapeutic agents can be incorporated for administration into a single tablet or other dosage form (e.g., in combination therapy); however, in other embodiments, the therapeutic agents can be provided in separate dosage forms.

[0168] Suitable inert materials include diluents such as carbohydrates, mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextran, starch, etc., or inorganic salts such as tricalcium phosphate, calcium phosphate, sodium phosphate, calcium carbonate, sodium carbonate, magnesium carbonate, and sodium chloride. Disintegrants or granulating agents may be included in the formulation, for example, starch (such as corn starch), alginic acid, sodium starch glycolate, Amberlite, sodium carboxymethyl cellulose, hyperbranched starch, sodium alginate, gelatin, orange peel, acidic carboxymethyl cellulose, natural sponge, and bentonite, insoluble cation exchange resins, powdered gums (such as agar, karaya, or alginic acid) or their salts.

[0169] Binders can be used to form hard tablets. Binders include materials from natural products such as gum arabic, starch, and gelatin, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl methyl cellulose, etc.

[0170] Lubricants may be included in the tablet formulation, such as stearic acid or its magnesium or calcium salts, polytetrafluoroethylene, liquid paraffin, vegetable oils and waxes, sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol, starch, talc, pyrogenic silica, hydrated silicoaluminate, etc.

[0171] Surfactants may also be employed, for example, anionic detergents (such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate), cationic detergents (such as benzalkonium chloride or benzethonium chloride), or nonionic detergents (such as polyoxyethylene hydrogenated castor oil, glycerol monostearate, polysorbate, sucrose fatty acid ester, methyl cellulose, or carboxymethyl cellulose).

[0172] Controlled-release formulations may be used, in which the active agent or its analog is incorporated into an inert matrix that allows release by diffusion or leaching mechanisms. Slowly degradable matrices may also be incorporated into the formulation. Other delivery systems may include timed-release, delayed-release, or sustained-release delivery systems.

[0173] Coatings may be used, for example, non-enteric materials (such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methyl hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, and polyethylene glycol) or enteric materials (such as phthalates). Dyes or pigments may be added to facilitate the identification or characterization of different combinations of the active agent dose.

[0174] When administered orally in liquid form, liquid carriers such as water, oils of animal or vegetable origin (such as peanut oil, mineral oil, soybean oil or sesame oil), or synthetic oils can be added to the active ingredient. Physiological saline solutions, glucose or other sugar solutions, or diols (such as ethylene glycol, propylene glycol or polyethylene glycol) are also suitable liquid carriers. The pharmaceutical composition can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil (such as olive oil or peanut oil), a mineral oil (such as liquid paraffin), or a mixture thereof. Suitable emulsifiers include naturally occurring gums (such as gum arabic), naturally occurring phospholipids (such as soy lecithin), esters or partial esters derived from fatty acids and hexitol anhydrides (such as sorbitan monooleate), and condensation products of these partial esters with ethylene oxide (such as polyoxyethylene sorbitan monooleate). The emulsion can also contain sweetening and flavoring agents.

[0175] Pulmonary delivery of the active agent can also be employed. The active agent can be delivered to the lungs upon inhalation and pass through the pulmonary epithelial lining to reach the bloodstream. A variety of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. These devices employ formulations suitable for dispensing the active agent. Generally, each formulation is specific to the type of device employed and can involve the use of appropriate propellant materials in addition to diluents, adjuvants, and / or carriers that can be used in therapy. The active ingredient can be prepared in particulate form for pulmonary delivery, with the particles having an average particle size of 0.1 μm or less up to 10 μm or greater, for example, about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 μm to about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5 μm. Pharmaceutically acceptable carriers for pulmonary delivery of the active agent include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for the formulation can include DPPC, DOPE, DSPC, and DOPC. Natural or synthetic surfactants can be used, including polyethylene glycol and dextrans such as cyclodextrin. Bile salts and other related enhancers, as well as cellulose and cellulose derivatives, and amino acids can also be used. Liposomes, microcapsules, microspheres, inclusion complexes, and other types of carriers can also be employed.

[0176] Drug formulations suitable for jet or ultrasonic nebulizers generally comprise an active agent dissolved or suspended in water at a concentration of from about 0.01 or less to 100 mg or more of active agent per mL of solution, e.g., from about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 mg per mL of solution. The formulation may also contain a buffer and a monosaccharide (e.g., for protein stabilization and osmotic pressure adjustment). Nebulizer formulations may also contain a surfactant to reduce or prevent surface-induced aggregation of the active agent caused by solution atomization during aerosol formation.

[0177] Formulations for use with metered dose inhaler devices generally comprise a finely divided powder containing an active ingredient suspended in a propellant with the aid of a surfactant. The propellant may comprise conventional propellants such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons and hydrocarbons. Exemplary propellants include trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, 1,1,1,2-tetrafluoroethane and combinations thereof. Suitable surfactants include sorbitan trioleate, soy lecithin and oleic acid.

[0178] Formulations for dispensing from a powder inhaler device generally comprise a finely divided dry powder containing an active agent, which optionally contains a filler such as lactose, sorbitol, sucrose, mannitol, trehalose or xylitol in an amount that facilitates dispersion of the powder from the device, generally from about 1 wt.% or less to 99 wt.% or more of the formulation, e.g., from about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 wt.% to about 55, 60, 65, 70, 75, 80, 85 or 90 wt.% of the formulation.

[0179] In some embodiments, the active agents provided herein may be administered intravenously, parenterally or by other injection in the form of a pyrogen-free, parenterally acceptable aqueous solution or oily suspension. Suspensions can be formulated using suitable dispersing or wetting agents and suspending agents in accordance with methods well known in the art. Preparation of acceptable aqueous solutions at suitable pH, isotonicity, stability, etc. is within the skill of the art. In some embodiments, the pharmaceutical composition for injection may contain an isotonic vehicle such as 1,3-butanediol, water, isotonic sodium chloride solution, Ringer's solution, glucose solution, glucose and sodium chloride solution, lactated Ringer's solution, or other vehicles known in the art. In addition, a sterile fixed oil is conventionally employed as a solvent or suspending medium. For this purpose, any mild fixed oil may be employed, including synthetic mono- or di-glycerides of fatty acids. In addition, fatty acids such as oleic acid may similarly be used to form injectable preparations. The pharmaceutical composition may also contain stabilizers, preservatives, buffers, antioxidants or other additives known to those skilled in the art.

[0180] The duration of the injection can be adjusted according to various factors and can include a single injection administered in a process of a few seconds or less to continuous intravenous administration for 0.5, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours or longer.

[0181] Dose

[0182] In one aspect, based on the weight of the composition, the medicaments disclosed herein can be present in an amount of from about 0.5% to about 95%, or from about 1% to about 90%, or from about 2% to about 85%, or from about 3% to about 80%, or from about 4%, about 75%, or from about 5% to about 70%, or from about 6%, about 65%, or from about 7% to about 60%, or from about 8% to about 55%, or from about 9% to about 50%, or from about 10% to about 40%.

[0183] In one aspect, the compound can be administered at a rate of 100 μg to 1000 mg / day / kg body weight. Orally, the compound can be administered at a rate of from about 100, 150, 200, 250, 300, 350, 400, 450 or 500 μg to about 1, 5, 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg / day / kg body weight. The desired dose can be administered in one or more portions. For oral administration, suitable forms are, for example, tablets, gels, aerosols, pills, dragees, syrups, suspensions, emulsions, solutions, powders and granules; one method of administration comprises using a suitable form containing from 1 mg to about 500 mg of the active substance. In one aspect, the administration can include using a suitable form containing from about 1, 2, 5, 10, 25 or 50 mg to about 100, 200, 300, 400, 500 mg of the active substance.

[0184] The dosing regimen will vary according to known factors such as the pharmacodynamic and pharmacokinetic properties of the agent, and their mode and route of administration; the species, age, sex, health status, medical condition and weight of the patient, the nature and degree of the symptoms, the type of concurrent treatment, the treatment frequency, the route of administration, the renal and hepatic function of the patient, and the desired effect. A skilled physician can readily determine the effective amount of the drug required to prevent, counteract or arrest the progression of the symptoms or effects of muscle contracture. In one aspect, the active agents provided herein can be administered in dosage forms selected from intravenous or subcutaneous unit dosage forms, oral, parenteral, intravenous and subcutaneous. In some embodiments, the active agents provided herein can be formulated as liquid preparations for, for example, oral administration. Suitable forms include suspensions, syrups, elixirs, etc. In some embodiments, unit dosage forms for oral administration include tablets and capsules. Unit dosage forms configured to be administered once daily; however, in certain embodiments, it may be desirable to configure the unit dosage form to be administered two or more times daily.

[0185] In some embodiments, the active agents provided herein can additionally employ auxiliary components conventionally present in pharmaceutical compositions, in a manner and at a level established in the art. Thus, for example, the composition can contain additional compatible pharmaceutically active materials for combination therapy or can contain materials for physically formulating various dosage forms, such as excipients, dyes, thickeners, stabilizers, preservatives or antioxidants.

[0186] In some embodiments, the active agents provided herein can be provided to the administering physician or other healthcare professional in the form of a kit. The kit is a package containing containers, the containers containing one or more active agents in a suitable pharmaceutical composition, and instructions for administering the pharmaceutical composition to a subject. The kit can also optionally contain one or more additional therapeutic agents currently used to treat a disease state such as described herein. For example, a kit can be provided containing one or more compositions comprising a combination of the active agent provided herein with one or more additional active agents, or a kit can be provided containing separate pharmaceutical compositions of the active agent provided herein and an additional therapeutic agent. The kit can also contain individual doses of the active agent provided herein for sequential or consecutive administration. The kit can optionally contain one or more diagnostic tools and instructions for use. The kit can contain suitable delivery devices, such as syringes, etc., and instructions for administering the active agent and any other therapeutic agent. The kit can optionally contain instructions for storage, reconstitution (if applicable) and administration of any or all of the therapeutic agents contained therein. The kit can contain multiple containers, which reflect the number of administrations to be given to the subject.

[0187] Examples

[0188] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. Those skilled in the art should understand that the techniques disclosed in the subsequent examples represent methods that have been found to function well in the practice of the invention and thus can be considered as examples of its practice patterns. However, based on the present disclosure, those skilled in the art should understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention and still obtain the same or similar results.

[0189] Ras is a target for several human cancers and a group of genetic diseases known as RASopathies (Aoki et al., 2016; McCormick, 2016; Simanshu et al., 2017; Tidyman and Rauen, 2009b). The applicant targeted the GTP-bound form of the G60A point mutant previously described by the applicant (Ford et al., 2005). The rationale for targeting the RasG60A structure by virtual screening is as follows. First, a potential binding site cleft located between switch 1 and the triphosphate nucleotide can be identified in this structure, although it is small. This cleft is due to the adoption of the switch 1 region in the GTP-bound structure (but not the GDP-bound structure) of this mutant. This conformation is different from wild-type Ras but similar to nucleotide-free Ras complexed with the guanine nucleotide exchange factor Sos (Boriack-Sjodin et al., 1998), which the applicant refers to as the "open conformation" of Ras. Switch 1 corresponds to residues 24-40 and is responsible for GTP- / Mg 2+ coordination as well as binding to effectors and regulators (Pai et al., 1989; Vetter and Wittinghofer, 2001; Wittinghofer and Nassar, 1996). Second, this mutation severely weakens the binding of Ras to its effector Raf kinase in vitro (Ford et al., 2005) and restores the transforming ability of constitutively active Ras in cells (Hwang et al., 1996; Sung et al., 1995; Sung et al., 1996). Third, previous use of solution 31P-NMR spectroscopy indicates that GTP-bound Ras adopts two conformations in equilibrium (Geyer et al., 1996; Spoerner et al., 2010; Spoerner et al., 2007): one conformation is capable of effector binding and thus signaling, while the other conformation is non-signaling and is mimicked by the G60A and T35S mutants (Araki et al., 2011; Shima et al., 2010; Spoerner et al., 2001). Taken together, it is thought that small molecules that keep Ras in an open conformation may inhibit its signaling. Previously, a similar approach led to the discovery of the anti-Ras "Kobe" compound (Shima et al., 2013).

[0190] Combined data from virtual screening, cell growth and colony formation assays, and chemical synthesis and analysis identified a small molecule called IODVA1 that has cytostatic activity against several transformed cell lines, including Ras-driven cells. The applicant shows that IODVA1 targets Rac activation and signaling. The applicant has demonstrated that IODVA1 has in vivo activity against human MDA-MB-231 triple-negative breast cancer (TNBC) and H2122 non-small cell lung cancer (NSCLC) cell lines in a xenograft mouse tumor model.

[0191] Results

[0192] Docking. Using the Autodock program (Huey et al., 2007; Morris et al., 1998; Morris et al., 2009), the applicant performed virtual screening of small molecules that might fit into the identified Ras-binding interface pocket. The pocket is arranged by Ile21, Gln22, switch 1 residues Gln25-Pro34, Lys147, and Arg149, and GTP-ribose ( Figure 1 A). Approximately 118,500 compounds from the NCI / DTP Open Chemical Repository were used to identify potential binders. The applicant designed a grid box containing the Ras pocket for docking compounds and searched for the grid box with the lowest possible binding energy. The predicted binding poses returned by Autodock were grouped in binding clusters with a root mean square deviation (rmsd) tolerance less than The results were evaluated by ranking the various complexes according to their predicted binding energies. Subsequently, clustering analysis was completed based on the rmsd values relative to the starting ligand geometries. The docking conformations with the most favorable binding free energies and larger clusters were selected as the best results. For each cluster, the estimated binding free energy in kcal / mol was obtained, and the estimated inhibition constant (Ki) at 298.15 K was derived. The compounds were classified according to their Ki. When obtained from the National Cancer Institute Developmental Therapeutics Program (NCI / DTP, http: / / dtp.nci.nih.gov), forty compounds with the highest Ki were requested and obtained. If possible, the compounds were dissolved in DMSO as 10 mM stock solutions, or at lower concentrations when solubility was poor.

[0193] Cell and biochemical phenotype screening assays. To evaluate the effect on proliferation, 40 hit compounds were tested in cell assays. Using the MTS cell proliferation assay, 10 μM of the hits were screened against the human lung mucoepidermoid carcinoma cell line H292 and the human lung adenocarcinoma cell line A549 (Berridge et al., 2005; Denizot and Lang, 1986; Loveland et al., 1992). These two epithelial cell lines were chosen because H292 encodes wild-type RAS (RAS WT ) while A549 encodes KRAS G12V mutations, with the expectation that compounds that distinguish between these two cell lines should be specific for carcinogenicity associated with RAS WT . Figure 1 Panel B shows that most compounds had little effect on this cell line (mean relative proliferation = 127.7% and STDEV = 36.0%), except that NSC600805 and NSC124205 significantly reduced the proliferation of A549 cells compared to the vehicle control; the proliferation rate of some compounds almost doubled. The average proliferation rates of NSC600805 and NSC124205 on A549 cells were 34.4% and 32.9%, respectively, corresponding to z-scores of 2.60 and 2.63 relative to the DMSO vehicle control. When tested on H292 cells, some compounds in the test group (mean compound relative proliferation = 88.3% and STDEV = 24.1%) reduced their proliferation by 40% or more, but our attempt to identify a compound with anti-proliferative effects on A549 but no or less effect on H292 cells failed. The H292 cell proliferation rates of NSC600805 and NSC124205 relative to the vehicle control were 49.4% and 41.6%, respectively, corresponding to z-scores of 1.61 and 1.94 ( Figure 1 Panel B).

[0194] Although 5 μM of NSC124205 inhibited the growth of single mutant yeast strains containing the rad50, mec2, and bub3 genes, as well as double mutants sgs1+mgt1, cln2+rad14, and mlh1+rad18 strains, it was still better than 80% compared to the untreated control. The results of the NCI screening were consistent with those shown here.

[0195] Biochemistry of NSC124205. The applicant tested the ability of NSC124205 and 7 other compounds to acutely reduce ERK and AKT phosphorylation in 3T3 cells. Cells were serum-starved for 24 hours, incubated with DMSO vehicle control or with 15 μM of the compound for 1 hour, stimulated with EGF for 5 minutes, and changes in ERK and AKT phosphorylation were examined by immunoblotting. Figure 2 A shows that NSC124205 significantly reduced both pERK and pAKT. To confirm that this reduction was not cell-specific, the applicant tested the effect of NSC124205 on the NF1-related malignant peripheral nerve sheath tumor (MPNST) cell line ST8814, which is characterized by active wild-type Ras (Basu et al., 1992; Mahller et al., 2006). ST8814 was grown in RPMI medium supplemented with 10% FBS for 24 hours and 48 hours in the presence or absence of 10 and 50 μM of NSC124205, and the levels of pAKT and pERK were quantified by immunoblotting. Figure 2 B shows that after 24 hours of incubation, 10 and 50 μM of NSC124205 reduced the level of pERK by at least 50%. For the 10 μM dose, the reduction in pERK remained unchanged at 48 hours, while for the 50 μM dose, it increased substantially. The pAKT level decreased in a time- and dose-dependent manner. Taken together, the cellular and biochemical data indicate that NSC124205 reduced the proliferation of Ras-driven cell transformation and reduced ERK phosphorylation, although at high concentrations.

[0196] To probe the effect of NSC124205 on oncogenic Ras signaling, the applicant generated NIH-3T3 cells overexpressing wild-type or dominant-active HRAS G12V ( Figure 2 C), and tested the ability of the compound to interfere with acute ERK activation in these cells. Cells were serum-starved for 24 hours, incubated with 10 μM or 50 μM of NSC124205 and two other compounds for 2 hours, and activated with EGF / serum for 5 minutes. Cells were lysed, normalized, and the cell lysates were separated by SDS-PAGE and blotted for pERK. In cells expressing RAS WT or RAS G12VIn the cells, pERK was not decreased in a NSC124205 dose-dependent manner ( Figure 2 C). Thus, the mechanism of action of NSC124205 is independent of Ras activating mutations.

[0197] Inhibition of anchorage-independent growth. Using a soft agar colony formation assay, NSC124205 was screened for its ability to inhibit the proliferation of 3T3 cells overexpressing HRAS at concentrations of 5 and 25 μM. As G12V shown, both NSC124205 concentrations reduced colony formation of 3T3 cells by 60%, with no significant difference between the two concentrations. Figure 2 D).

[0198] Chemical synthesis of IODVA1. Prior to in vivo studies, the applicant examined the identity and purity of compound NSC124205. The applicant tested a freshly prepared DMSO solution of NSC124205 obtained from the NCI by high performance liquid chromatography-mass spectrometry (HPLC / MS). As Figure 3 shown in A, the HPLC curve of the NSC124205 solution was consistent with a mixture of at least three components that absorb at 210 nm and elute at approximately a 2:1:1 ratio at 11.6 minutes, 12.8 minutes, and 13.4 minutes, respectively. Examination of the UV curves of each peak showed that the peaks at 12.8 minutes and 13.4 minutes had similar absorbance curves with maxima close to 250 nm and 300 nm. The mixture was analyzed by LCMS using the same column and conditions Figure 3 F). The first band (peak 1a) at 11.6 minutes had a mass-to-charge ratio m / z of 264.11178 [M+2H + and 527.21607 [M+H + , and an elemental composition of C 28 H 23 N 12 + that equaled the exact mass of 527.21632 Da. This band likely corresponds to two guanidinobenzimidazole additives ( Figure 3 F and 3I). This is consistent with the observation that its spectrum lacks a red-shifted absorbance maximum due to the lack of an extended aromatic system. The other two bands at 12.8 and 13.4 minutes had the same m / z 370.14096 [M'] and elemental composition C 20 H 16 N7O + that equaled the exact mass of 370.14108 Da. The small peak at m / z 392.12286 corresponded to [M'+Na +. Both bands have similar absorbance spectra, indicating a similar structural basis and possible tautomers. Neither peak gave a mass corresponding to the structure of NSC124205 reported on the PubChem website ( Figure 1 B), whose calculated mass is 352.1305 Da. A mass difference of 18 Da indicates the presence of an additional (O + 2x H) in the structure of NSC124205.

[0199] The applicant is concerned with Figure 1 the structure shown in B and reported on the PubChem site. The applicant synthesized a compound with the following structure, hereinafter referred to as IODVA1:

[0200]

[0201] (IODVA1): The synthesis was derived from similar molecules in the literature. 2-Guanidino benzimidazole and α-pyridine were heated at 90 °C in the presence of acetic acid. HPLC analysis of the synthesized compound showed a single band observed at 12.8 minutes, which corresponded to peak 1b observed in the NSC124205 sample ( Figure 3 A and 3F). The UV curve of the synthesized compound also matched peak 1b of the provided sample. MS analysis of the synthesized molecule showed a mass of 370.1409 [M+H + , and MS-MS data indicated that the additional 18 Da mass we saw in the NCI sample was covalently bonded to the molecule ( Figure 3 G). Two molecules with the same exact mass as the synthesized molecule could be generated from the synthetic route ( Figure 3 C and 3F). Molecule (A) is the result of the condensation of 2-guanidino benzimidazole and the 2-2'-pyridine ring. Molecule (B) is the result of a pinacol-like rearrangement of (A). H 1 -NMR analysis ( Figure 3 G) showed that there were 13 protons in the aromatic region consistent with structure (A), and there was no evidence of symmetry indicating structure (B). This was also confirmed by C 13 -NMR and infrared (IR) spectroscopic analyses, which showed no peak near 170 - 190 ppm and no stretching near 1680 cm -1 respectively, which are characteristic of a carbonyl group ( Figure 3 D and 3E). Thus, the spectral data is consistent with IODVA1 (the synthesized molecule, i.e., structure (A)).

[0202] Analysis of IODVA1 showed that despite its high nitrogen content and aromaticity, it has some favorable drug-like properties. The applicant performed several calculations to predict the druggability of IODVA1. The calculations showed that the target molecule is non-planar, reasonably soluble in the aqueous phase, has a logP of 3.25, one hydrogen bond donor, and four hydrogen bond acceptors. Due to these chemical properties, IODVA1 follows Lipinski's rule of five.

[0203] Effect of IODVA1 on the proliferation of cells containing activated Ras. The applicant tested the ability of IODVA1 to inhibit the proliferation of cells containing activated Ras and thus its ability to recapitulate the compounds obtained from the NCI. The applicant selected the NF1-related malignant peripheral nerve sheath tumor (MPNST) cell line ST8814, which is characterized by active wild-type Ras (Basu et al., 1992; Mahller et al., 2006) and the triple-negative breast cancer cell line MDA-MB-231 carrying the oncogenic KRASG13D mutation (Hollestelle et al., 2007). In addition, the applicant used the untransformed mammary epithelial cell line MCF10A (RAS WT Immortalized by spontaneous t(3;9)(3p13;9p22) translocation that deletes the CDKN2A gene, also known as p16), and the non-invasive estrogen receptor (ER) and progesterone receptor (PR) positive RASWT breast cancer cell lines MCF7 (wild-type p53) and T47D (mutant p53). Cells were grown in growth medium containing IODVA1 (at concentrations between 0.1 and 10 μM) or vehicle control, and cell numbers were counted using trypan blue exclusion. Figure 4 D shows that increasing concentrations of IODVA1 inhibited the proliferation of ST8814 cells, with a growth inhibitory concentration (GI50) of 50% at 1 μM on day 4. Similar results were observed in MCF7, MDA-MB-231, and T47D cells, with an estimated GI50 ≤ 1 μM ( Figure 4 A). No significant decrease in the proliferation of untransformed MCF10A cells was observed. At 1 and 3 μM, IODVA1 significantly reduced the number of colonies of breast cancer cells in soft agar, which is consistent with the cell proliferation results ( Figure 4 B).

[0204] IODVA1 and Ras activation. To examine whether IODVA1 inhibits Ras activation in cells, the Applicant determined the levels of active GTP-bound Ras in ST88-14 cells treated with IODVA1 (2 μM) or vehicle control at different time points. Glutathione beads conjugated with GST-RafRBD were incubated with ST8814 cell lysates, washed thoroughly, and the protein complexes were separated on SDS-PAGE. The levels of GTP-bound pan-RAS proteins that bind to RafRBD were determined by immunoblotting. As Figure 4 shown in C, the levels of active Ras were similar between IODVA1- and vehicle control-treated cells after 24 hours of treatment. However, a decrease in the level of active Ras was seen after 48 hours of drug treatment, and was very evident at the 72-hour time point. Without being bound by theory, since it takes at least 48 hours for the level of active Ras to decrease, this may be because IODVA1 does not bind to Ras and its mechanism of action is independent of Ras.

[0205] IODVA1 interferes with the formation of lamellipodia and circular dorsal ruffles. The progression of cancer invasion and metastasis requires the abnormal activation of cell migration, which is driven by the reorganization of the actin cytoskeleton (Condeelis et al., 2005; Sahai, 2005; Yamaguchi and Condeelis, 2007; Yamaguchi et al., 2005). A major feature of Ras-transformed cells is the reorganized actin cytoskeleton, which results in poor adhesion, increased motility, invasiveness, and non-contact growth. To examine the effect of IODVA1 on the overall actin cytoskeleton structure, MDA-MB-231 cells were serum-starved for 4 hours, treated with IODVA1 (0 - 3 μΜ) for 1 hour, and then the cells were stimulated with EGF for 30 minutes, which induces the formation of lamellipodia. Figure 5 A shows that compared to vehicle-treated cells (0 μM), IODVA1 treatment inhibited the formation of lamellipodia and the enrichment of cortical filamentous actin. Cells treated with 1 and 3 μM IODVA1 had more prominent stress fibers and a rounded cell shape (open arrows). In parallel experiments, the actin cytoskeleton structure of cells stimulated with EGF was examined, then washed and subsequently treated with IODVA1 or vehicle control. Figure 5 E shows that within 30 minutes after treatment with IODVA1 (3 μM), the overall filamentous actin staining intensity decreased.

[0206] Lamellipodia formation and membrane ruffling in response to growth factor stimulation are characteristic of Rac activation (Ridley and Hall, 1992; Ridley et al., 1992). Based on the observation that IODVA1 treatment impeded lamellipodia formation, the applicant evaluated the effect of IODVA1 on another Rac-mediated actin structure - circular dorsal ruffles (CDRs) (Steffen et al., 2013). CDRs are closed, dynamic, circular structures that stand vertically and appear on the dorsal side of the cell, and their formation requires Rac activity. 3T3 fibroblasts were starved for 4 hours, treated with IODVA1 (0–3 μM) for 1 hour, and stimulated with PDGF for 10 minutes. Figure 5 Panel B shows that CDR formation was inhibited by IODVA1 treatment. Cells treated with 1 and 3 μM IODVA1 had intact stress fibers, exhibited a "starfish" or triangular cell shape and had no protrusions.

[0207] IODVA1 inhibits Rac activation. To confirm that IODVA1 interferes with Rac activation, the applicant examined the levels of active Rac and its downstream effector PAK1 / 2. MDA-MB-231 cells were incubated with IODVA1 (0–3 μM) for 1 hour, and the levels of GTP-bound active Rac were measured by GST-PAK-GBD pull-down and quantified ( Figure 5 Panel C). IODVA1 significantly reduced the levels of active Rac in a dose-dependent manner, and the levels of the related active Cdc42 GTPase were also reduced, but only at the highest IODVA1 concentration. The levels of active RhoA were not affected by IODVA1 ( Figure 5 Panel C), and no effect on stress fiber alignment was observed. Similarly, MDA-MB-231 cells incubated with IODVA1 (1 μM) for 30 minutes experienced a 50% reduction in the levels of pPAK1 / 2 (T423 / T402). However, the levels of pPAK4 / 5 / 6 did not change even after 3 hours of incubation; PAK4 / 5 / 6 is mainly Cdc42-specific ( Figure 5 Panel D). Collectively, these data indicate that at low concentrations, IODVA1 inhibits Rac activation and downstream signaling, thereby inhibiting lamellipodia and CDR formation.

[0208] IODVA1 can reduce cell-ECM and cell-cell interactions. The applicant evaluated whether IODVA1 interferes with cell spreading on the extracellular matrix, an event that is also controlled by rearrangement of the actin cytoskeleton. MCF7, T47D or MDA-MB-231 cells were plated on fibronectin-coated coverslips for 10 minutes, incubated for an additional 30 minutes in the presence of 0 - 3 μM IODVA1, fixed, and examined by bright-field microscopy.Figure 6 Panel A shows that exposure to IODVA1 interferes with the spreading of MCF7 (0.3 μM) and MDA-MB-231 (1 μM), as indicated by the decrease in cell area. Although more rounded cells were observed in the presence of IODVA1, no change in the area of T47D cells was detected. These results suggest that upon contact with the extracellular matrix and mitogen stimulation, cells treated with IODVA1 are unable to initiate and / or maintain the actin reorganization required for cell spreading and leading edge formation.

[0209] In vitro 3D assays, such as spheroid formation, act as an intermediate between 2D (monolayer) cell assays and in vivo animal models. Spheroid formation is mediated by matrix development and remodeling, as well as changes in the cytoskeleton and cell-cell contact and adhesion. To evaluate the effect of IODVA1 on spheroid formation, single cell suspensions were plated in complete medium containing vehicle control or IODVA1 (ranging from 0.1 - 3 μM) using the hanging drop and ultra-low attachment (ULA) methods (Foty R. 2011).

[0210] In the hanging drop method, spheroids were mixed with the indicated concentration of IODVA1 in complete medium and then formed as 25 μL hanging drops on the lid of a 10-cm culture dish. After 96 hours, the spheroids were transferred to a 10-cm culture dish using a wide pipette tip and imaged using bright-field microscopy before and after trituration. Figure 6 Panel B shows that IODVA1-treated MCF7 cells formed smaller spheroids at 1 μM. Mechanical disruption caused by pipetting led to complete dissociation of the spheroids at 1 μM IODVA1. T47D cells treated with 1 μM IODVA1 failed to form aggregated spheroids and instead remained as cell aggregates. MDA-MB-231 formed loose aggregates rather than tight spheroids, which were not affected by IODVA1 treatment but decomposed into smaller aggregates after trituration. No effect was observed in IODVA1-treated MCF10A cells.

[0211] To form spheroids in ultra-low attachment plates, MCF7, T47D, and MDA-MB-231 (5,000 cells) were incubated in complete medium with IODVA1 (0 - 3 μM) for 5 days. The aggregates and spheroids were dissociated with Accutase and live cells were counted by trypan blue exclusion. Treatment with IODVA1 significantly reduced the number of live cells in the spheroids ( Figure 6 Panel C). These results suggest that IODVA1 may effectively inhibit spheroid formation in 3D culture systems by suppressing proliferation.

[0212] IODVA1 kinase inhibitory activity. IODVA1 has two pyridine groups attached to the central imidazole group. Pyridine is one of the most common scaffolds in kinase inhibitors (Xing et al., 2014), and is present in several potent inhibitors of FLT3, Aurora, ROCK, AKT, and other kinases (Bavetsias et al., 2012; Green et al., 2015; Woods et al., 2006), suggesting that IODVA1 may possess kinase inhibitory activity. To test this hypothesis, the applicant evaluated the potential of IODVA1 to interfere with the ability of 369 recombinant wild-type kinases to hydrolyze ATP. Each kinase was tested twice at a single IODVA1 concentration of 0.5 μM, and the data were averaged and compared to a vehicle DMSO control. Figure 7 Plots are shown comparing replicates to a vehicle control set at 100%. Statistical analysis of the kinase panel data indicated that IODVA1 was inactive in vitro against 98.6% (364 out of 369) of the tested kinases. It showed reduced activity (22% to 27%) of ACK1, TSSK3 / STK22C, GSK3b, and IRAK1, while the activity of YSK4 / MAP3K19 was increased (21%), greater than 3 standard deviations (>3σ). However, the inhibitory and stimulatory effects were modest, and higher concentrations of IODVA1 were required to inhibit or stimulate the above kinases to the 50% level. Therefore, IODVA1 is not a kinase inhibitor, and given that the cellular GI50 of IODVA1 (0.5 to 1 μM) is similar to the concentration used in the kinase assay, the previously observed cellular effects cannot be explained by kinase inhibition or stimulation.

[0213] IODVA1 reduces tumor burden of solid tumors in vivo. To examine whether the ability of IODVA1 to reduce oncogene-driven cell proliferation can be translated in vivo, the applicant tested its efficacy in a breast cancer and a lung cancer xenograft mouse model. The breast cancer model utilized human triple-negative breast cancer MDA-MB-231 cells. Cells were injected orthotopically into the left and right inguinal mammary glands of immunodeficient female mice. Then, tumor-bearing mice received intraperitoneal (IP) injections of 250 μL of 1 mM IODVA1 every other day, with an average dose of 3.5 mg / kg. The dosing regimen was not optimized, and it was expected that better results could be obtained by increasing the IODVA1 dose. Compared to vehicle-treated control mice, a significant reduction in tumor volume (≥50%) was observed four weeks after treatment ( Figure 8 A-8C). While the tumor volume of vehicle-treated mice doubled, the tumors treated with IODVA1 did not exceed the tumor size before treatment ( Figure 8 A). The tumors were then excised, fixed, and paraffin-embedded, and then stained immunohistochemically for the proliferation marker (Ki-67), the apoptosis marker (cleaved caspase-3), and with DAPIFigure 8 B). Comparison of Ki-67 stained tumors treated with vehicle control and IODVA1 did not reveal statistical changes in cell proliferation. However, tumor sections stained with cleaved caspase-3 showed a significant increase in apoptosis in cells treated with IODVA1 compared to vehicle control ( Figure 8 C) Therefore, IODVA1 has the ability to induce caspase activation, thereby limiting tumor growth in vivo.

[0214] At the end of the four-week treatment, mice were euthanized and peripheral blood was collected by cardiac puncture to examine the effects of IODVA1 on the white and red blood cells of treated mice for any signs of toxicity. There were no significant differences in WBC, neutrophils, lymphocytes, monocytes, RBC, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin volume (MCHV), red cell distribution width (RDW), platelets, and mean platelet volume between the blood of drug- and vehicle-treated mice ( Figure 9 ).

[0215] The lung cancer mouse model was generated by injecting lung cancer H2122 cells into the right and left sides of NSG mice. These cells carry KRAS G12C mutations and formed aggressive tumors. Mice were treated with IODVA1 or vehicle control every other day for 14 days. Here, a significant reduction in tumor volume was also observed in mice treated with IODVA1 ( Figure 8 D). Tumors were excised and sections were stained with hematoxylin and eosin (H&E) and for the Ki67 proliferation marker ( Figure 8 E).

[0216] Vehicle-treated H2122 tumors were filled with tumor cells with a high mitotic rate ( Figure 8 Arrows in E). IODVA1-treated H2122 tumors showed a decrease in mitotic cell frequency and increased stromal cell infiltration, indicative of a treatment-induced fibrotic response. Quantification of Ki67-positive cells showed a significant decrease in the number of Ki67-positive tumor cells in mice treated with IODVA1 ( Figure 8 E), indicating that IODVA1 negatively affects cell proliferation in vivo.

[0217] In summary, our in vivo data suggest that IODVA1 is effective in treating solid tumors, including Ras-driven solid tumors, possibly by increasing tumor cell apoptosis and reducing cell proliferation. In addition, IODVA1 administration does not adversely affect bone marrow function due to the lack of biologically relevant changes in peripheral blood cell counts.

[0218] Ras lies at the center of many protein–protein interactions that are crucial for proper cell signaling and normal physiology. Loss of regulation by single point mutations or otherwise fine-tuning leads to reduced GTPase activity and is frequently present in cancers and a class of diseases called RASopathies (Rauen, 2013; Stephen et al., 2014; Tajan et al., 2018; Tidyman and Rauen, 2009a). Despite decades of long-term efforts by academic and private pharmaceutical companies, translating Ras targeting into a therapy has been extremely challenging. Excellent reviews discussing the subject and various approaches taken against Ras have been reported in the literature (Marin-Ramos et al., 2018; Ostrem and Shokat, 2016; Spencer-Smith and O'Bryan, 2017; Spiegel et al., 2014). High-resolution structures do not show deep druggable cavities, and no known Ras agonists or antagonists are available to provide a starting scaffold for building the chemical discovery process. Despite these difficulties, several low molecular weight chemicals have been reported to bind to Ras with low affinity (Cox et al., 2014; Maurer et al., 2012; Quevedo et al., 2018; Shima et al., 2013; Spiegel et al., 2014; Sun et al., 2012). Inhibiting the binding of Ras to its downstream effectors has also been challenging because Ras forms flat and large interfaces with Raf kinase, PI-3 kinase, and RalGDS The said RalGDS is formed by the juxtaposition of β-strands from each partner protein. Since GTP loading is such an important step in Ras signaling, recent efforts have aimed to interfere with GDP–GTP exchange by targeting the Ras / Sos interface (Evelyn et al., 2015; Evelyn et al., 2014; Maurer et al., 2012; Schopel et al., 2013; Spiegel et al., 2014; Sun et al., 2012). In general, whether these small molecules are effective in in vivo models of oncogenic Ras, whether their true mechanism of action is by directly inhibiting Ras, or whether they represent molecular scaffolds from which more effective molecules can be derived remains to be seen. If this task may seem impossible, recent reports suggest that it is not. For example, the discovery that compound 3144 binds to all Ras isoforms and shows anti-tumor activity in xenograft mouse cancer models is encouraging (Welsch et al., 2017). Covalent inhibition of KRAS in vivo G12C (a common mutation in lung adenocarcinoma) has also made similar progress (Janes et al., 2018).

[0219] In order to try to utilize the GTP-bound form of Ras G60A The cavity was identified in the crystal structure of Mg(Ford et al., 2005; Ford et al., 2009) and targeted by a small molecule that restricts the switch 1 loop to coordinate the Mg 2+ ions and using signaling conformations, the applicants were able to combine computer screening with cell proliferation and colony formation assays and identify NSC124205, a proliferation inhibitor of Ras-transformed cells at low micromolar concentrations. However, HPLC / MS analysis showed that NSC124205 was a mixture of at least three components. Other researchers have reported that a large proportion of commercially available chemicals fail quality control by UPLC (Corsello et al., 2017). This prompted the applicants to identify the active ingredient responsible for the antiproliferative activity of the NSC124205 mixture. After attempting a simple synthetic scheme to re-synthesize NSC124205, the applicants synthesized a novel small molecule with drug-like properties, which we call IODVA1. HPLC and MS analysis showed that IODVA1 corresponds to peak 1b of the NSC mixture. Considering that peak 1c is a tautomer of 1b, and considering that peak 1a is inactive in the cell assay, IODVA1 is considered to be the active ingredient in NSC124205.

[0220] RAS expression WT The high sensitivity of MCF7 and T47D cells, as well as MDA-MB-231 cells expressing oncogenic Ras, to IODVA1 suggests that IODVA1 targets nodes that Ras uses to regulate cell survival and cell spreading and motility, including lamellipodia formation. One such node is the small GTPase Rac. Biochemical observations that IODVA1 reduces Rac activation and signaling, such as PAK1 activity ( Figure 5 C and 5D) suggest that IODVA1 targets Rac signaling. Cellular and in vivo data support this idea. For example, IODVA1 inhibits the formation of Rac-driven cellular actin superstructures (including lamellipodia and CDRs) ( Figure 5 A and 5B) and affect cell spreading and shape within minutes of cell exposure ( Figure 6 A) and cell-cell adhesion ( Figure 6The ability of IODVA1 to target Rac activity is consistent with the observation that it reduces the levels of cleaved caspase-3 and proliferation in MDA-MB-231 and H2122 xenograft tumors in vivo, respectively, indicating that IODVA1 targets nodes required for Ras-regulated cell survival and proliferation, such as Rac. The role of Rac in regulating actin cytoskeleton organization has long been recognized (Etienne-Mannerville and Hall, 2002; Hall A., 1994; Lee and Dominguez, 2010). The role of Rac in regulating Ras-driven tumorigenesis in vivo has been reported in various mouse models (Mack et al., 2011; Kissil et al., 2007; Espina et al., 2008; Malliri et al., 2002; Wang et al., 2010; Bar-Sagi and Hall, 2000). IODVA1 appears to be specific for Rac, as it affects Cdc42, but only at high concentrations, and has no effect on Rho. This specificity is consistent with the inability of IODVA1 to reduce the levels of pPAK4 / 5 / 6 downstream of Cdc42 or to remodel stress fibers downstream of Rho.

[0221] In summary, the applicant has identified dipyridinium guanidinobenzimidazole derivatives with in vitro and in vivo activity against cancer cell line xenograft models. IODVA1 significantly reduces the proliferation of multiple cancer model cells with different genetic lesions, and it also functions at low micromolar concentrations. In vivo data show that it inhibits tumor growth by increasing apoptosis and / or reducing the proliferation of cancer cells. Cellular studies and studies of the peripheral blood of mice treated with IODVA1 for four weeks (12 doses) showed no significant toxicity and indicated that IODVA1 may be specific for transformed cells. The applicant's cellular and in vivo data are consistent with IODVA1 targeting Rac signaling.

[0222] Experimental procedures

[0223] Plasmids, cell lines, and reagents. MDA-MB-231, MCF7, T47D, MCF10A, and HEK293T cells were obtained from ATCC and have been authenticated by the DNA Diagnostic Center (Fairfield, OH) during the course of these studies. NIH-3T3 fibroblasts were a gift from Dr. Susanne I. Wells, ST8814 cells were a gift from Dr. Nancy Ratner, and A549 and H292 cells were a gift from Dr. Jeffrey Whitsett. MDA-MB-231 cells were maintained in modified MEM medium (Invitrogen) supplemented with 10% FBS, 1% penicillin / streptomycin, and 1% amphotericin B. MCF10A was maintained in DMEM / F12 (Invitrogen) supplemented with 5% horse serum, 20 ng / mL EGF, 0.5 mg / mL hydrocortisone, 100 ng / mL cholera toxin, and 10 μg / mL insulin. MCF7 and T47D were maintained in DMEM supplemented with 10% FBS and 10 μg / mL insulin. HEK293T and NIH / 3T3 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. ST8814, A549, and H292 cells were grown in RPMI supplemented with 10% FBS. Cells were treated with the indicated concentrations of compounds. Control cells were treated with only an equal volume of diluent. The following antibodies were used – GAPDH (GeneTex GTX627408, 1:5000), pERK1 / 2 T202 / Y204 (CST 4370, 1:2000), total ERK1 / 2 (CST 4696, 1:2000), pAKT S473 (9271, 1:1000), pPAK1 T423 / pPAK2 T402 (CST 2601S, 1:1000), PAK1 / 2 / 3 (CST 2604, 1:2000), PAK1 (CST 2602, 1:1000), pPAK4 S474 / pPAK5 S602 / pPAK6 S560 (CST3241, 1:1000), PAK4 (CST 62690, 1:1000), anti-mouse Eu (Molecular Devices R8205), anti-rabbit Eu (Molecular Devices R8204), anti-mouse HRP (CST 7076, 1:5000), and anti-rabbit HRP (CST7074, 1:5000), Ki67 (Abcam, IHC 1:100), cleaved caspase 3 (Asp175, CST 9661, IHC 1:100), goat anti-rabbit Alexa Fluor 568 (Abcam, IF 1:500).Fluorescent phalloidin and DAPI were from Invitrogen.

[0224] Computational virtual screening. Virtual screening was performed to identify candidate molecules that stabilize the Ras open conformation by targeting the cleft between the triphosphate nucleotide in the crystal structures of switch 1 and HRasG60A (PDB ID: 1XCM) Figure 1 A), where position 60 was reverted to Gly. The docking simulations for virtual screening were carried out using rigid body docking as implemented in AutoDock version 4.2 (Morris et al., 2009), in combination with the computational pipeline at the Protein Informatics Core of Cincinnati Children's Hospital Medical Center (CCHMC) on a Linux cluster with up to 512 CPUs. Polyview-3D (http: / / polyview.cchmc.org) was used to analyze the protein structure and guide the selection of the simulation box.

[0225] A subset of 118,500 drug-like synthetic compounds from the NCI / DTP Open Chemical Repository (http: / / dtp.cancer.gov) was used for virtual screening. These compounds were from NCI Plated 2007 stored in the Zinc database (http: / / zinc.docking.org / catalogs / ncip) by using chemical information filters (Irwin and Shoichet, 2005; Irwin et al., 2012; OpenEye Scientific Software). The 3D structures of the generated 118,500 compound subset were downloaded from ZINC. Gesteiger partial charges were used for Ras and chemical compounds. The screening was carried out in three stages, using increasingly stringent parameters and gradually expanding sampling ranges. The latter was achieved by increasing the number of energy evaluations (from 2x10 5 to 1x10 7 ), genetic algorithm runs (from 20 to 50), and population sizes (from 75 to 150), as described previously (Biesiada et al., 2011). After a preliminary quick screening, the top 30,000 candidates with the highest estimated binding affinities were retained and then re-scored using improved sampling in the refinement phase. Then the top 3,000 hits were re-scored using expanded sampling and further evaluated to select candidates for experimental validation.

[0226] These candidate compounds were ranked based on their estimated binding affinities and the entropy of the docking poses in multiple docking simulations, resulting in a set of 299 NCI library hits with an entropy of the docking poses lower than 0.2 and a predicted median binding constant less than 10 μM. Subsequently, Chemmine (http: / / chemmine.ucr.edu) was used to cluster these top hits based on their chemical similarity to further select candidates for experimental validation, while avoiding overrepresentation of certain classes of chemicals and visually analyzing the candidate compounds.

[0227] From this combined set, a subset of 40 compounds representing different chemical clusters was selected for experimental screening based on an assessment of drug-like properties, similarity to compound classes often identified as false positives in virtual screening, and the availability of compounds from the NCI / DTP Open Chemical Repository (http: / / dtp.cancer.gov).

[0228] MTS assay. The colorimetric CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS, Promega) was used to determine the number of viable cells and to evaluate the effect of the compounds on cell proliferation. Measurements were performed according to the supplier's protocol. The assay was carried out by directly adding 10 μL of the MTS reagent to the culture wells and then incubating for 1 h at 37 °C. The amount of formazan obtained at the end of the incubation was measured by absorbance at 490 nm using a 96-well plate reader (Molecular Devices; Sunnyvale, CA). Each 96-well plate had a set of four wells containing only medium and a set of four wells containing cells treated with DMSO vehicle control. The background absorbance was first evaluated from the set of wells containing only medium, averaged, and then subtracted from each well. The background-corrected absorbance readings were then normalized and expressed as a relative percentage of the plate-average DMSO vehicle control. Each experiment was repeated twice for each cell line, and in different experiments, the compounds were arranged randomly in the plates.

[0229] Chemical synthesis. All chemicals, reagents, and solvents were purchased from Sigma-Aldrich, Ark Pharm Inc., and Fisher Scientific. The specified reaction temperature refers to the temperature of the reaction bath, while room temperature (RT) is expressed as 25 °C. Analytical thin-layer chromatography (TLC) was performed using glass-backed silica plates (20 x 20 cm, pH = 5, MF254). Visualization was completed using a 254 nm UV lamp. 1H- and 1 H- and 1313C-NMR spectra. Chemical shifts are reported in ppm using tetramethylsilane as the standard. The data are reported as follows: chemical shift, number of protons, multiplicity (s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, b = broad, m = multiplet). All compounds were characterized by 1 1H-NMR, 13 13C-NMR and high-resolution mass spectrometry (HRMS).

[0230]

[0231] Scheme 1: Synthesis of IODVA1

[0232] (Z)-2-((1H-benzo[d]imidazol-2-yl)imino)-4,5-di(pyridin-2-yl)-2,5-dihydro-1H-imidazol-5-ol (IODVA1)

[0233] 2-Guanidinobenzimidazole (500 mg, 2.86 mmol) and α-pyridine (1.22 g, 5.72 mmol) were dissolved in N-N-dimethylformamide (5 mL). After adding glacial acetic acid (0.2 ml, 3.4 mmol), the reaction was stirred at 85 °C for 48 h. The reaction was cooled and then quenched with water, the pH was neutralized, and the aqueous solution was extracted three times with ethyl acetate. The combined organic layers were washed with water and brine, then dried over Na2SO4 and filtered using filter paper. Silica (2 g) was added and then the solution was concentrated under reduced pressure. The solid was loaded onto a chromatography column and then purified by flash chromatography using a 1:10 methanol:dichloromethane gradient. The target fraction (Rf = 0.5) was collected and concentrated under reduced pressure to give 111 mg (0.30 mmol, 11%) of the desired product as a brown-gold solid.

[0234] 1 1H NMR (400 MHz, MeOD-d6): δ = 7.07 (m, 3H), 7.33 (m, 2H), 7.51 (m, 2H), 7.63 (m, 1H), 7.85 (m, 1H), 8.05 (m, 1H), 8.19 (m, 1H), 8.33 (m, 1H), 8.53 (d, 1H).

[0235] 13 13C NMR (400 MHz, MeOD-d6): δ = 113.89, 122.04, 122.19, 123.16, 124.17, 124.74, 128.85, 129.53, 137.93, 138.93, 148.65, 149.81, 150.36, 150.60, 152.89, 156.43, 157.79, 161.23.

[0236] HRMS-ESI: [M+H] + (C 20 H 16 (C 20 H 16 N7O): Calculated value: m / z = 370.1411. Found value: m / z = 370.1409

[0237] Synthesis of NIRA2:

[0238] 2-((1H-Benzimidazol-2-yl)imino)-5,5-bis(pyridin-2-yl)imidazolidin-4-one and 2,2,5-tris(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2):

[0239]

[0240] 1,2-Bis(pyridin-2-yl)ethane-1,2-dione (5 g, 23.6 mmol) and 2-(1H-benzimidazol-2-yl)guanidine (5.37 g, 30.6 mmol) in DMSO (80 mL) were heated to 110 °C. 1 M aqueous KOH solution (28.3 mL) was added dropwise, and the mixture was maintained at 110 °C for 30 minutes. The mixture was poured into water (300 mL) and acidified to pH = 6 with 1 N HCl solution. The mixture was extracted with EtOAc (3 x 100 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was triturated with EtOH / DMF (5:1, 50 mL) and filtered to obtain 1 g of 2-((1H-benzimidazol-2-yl)imino)-5,5-bis(pyridin-2-yl)imidazolidin-4-one as a pale yellow solid and 0.72 g of 2,2,5-tris(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one. The two were further purified by reverse-phase HPLC (column: Kromasil C18 (250 * 50 mm * 10 um); mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 15%-40%, 10 minutes), to obtain 2,2,5-tris(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2) (100 mg, 1% yield). LCMS: m / z found value: 459.1 [M+H] +

[0241] 2,2,5-Tris(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2_P1) and 2,2,5-tris(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2_P2):

[0242]

[0243] 100 mg of the enantiomeric mixture was separated by SFC (column: DAICEL CHIRALCEL OJ (250 mm * 30 mm, 10 μm); mobile phase: [0.1% NH3H2O IPA]; B%: 43% - 43%, minutes), to obtain 22,25,25-tris(2-pyridyl)-29,30,31,32,33-pentaazatetracyclo[8.1.9.16,21(29),24(31)]pentacosa-23-one (NIRA2_P1) as a white solid (the faster eluting enantiomer, 35.75 mg, 36% yield) and 22,25,25-tris(2-pyridyl)-29,30,31,32,33-pentaazatetracyclo[8.1.9.16,21(29),24(31)]pentacosa-23-one (NIRA2_P2) as a white solid (the slower eluting enantiomer, 37.01 mg, 37% yield).

[0244] 2,2,5-Tris(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2_P1): MS: m / z found: 459.2 [M+H] + ; HPLC: Rt: 1.751 minutes; SFC: Rt = 1.734, on OJ column; 1 1H NMR (400 MHz, MeOD): δ 8.62 (d, J = 4.8 Hz, 1H), 8.50 (d, J = 4.8 Hz, 1H), 8.42 (d, J = 4.8 Hz, 1H), 7.86 - 7.84 (m, 4H), 7.51 - 7.41 (m, 5H), 7.36 - 7.28 (m, 2H), 7.20 (d, J = 8.0 Hz, 1H), 7.10 (t, J = 7.6 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H)

[0245] 2,2,5-Tris(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2_P2): MS: m / z found: 459.2 [M+H] + ; HPLC: Rt: 1.757 min; SFC: Rt = 1.893 min, on OJ column; 1 H NMR (400 MHz, MeOD): δ 8.62 (d, J = 4.8 Hz, 1H), 8.50 (d, J = 4.8 Hz, 1H), 8.42 (d, J = 4.8 Hz, 1H), 7.87 - 7.84 (m, 4H), 7.52 - 7.41 (m, 5H), 7.36 - 7.28 (m, 2H), 7.20 (d, J = 8.0 Hz, 1H), 7.10 (t, J = 7.6 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H).

[0246] Alternative synthetic route for NIRA2:

[0247]

[0248] 4-(Pyridin-2-yl)-3,4-dihydrobenzo[4,5]imidazo[1,2-a][1,3,5]triazin-2-amine:

[0249]

[0250] Under N2, piperidine (0.8 mL, 7.99 mmol) was added to a mixture of 2-(1H-benzo[d]imidazol-2-yl)guanidine (2 g, 11.4 mmol) and pyridine-2-carbaldehyde (1.83 g, 17.1 mmol) in EtOH (40 mL). The mixture was stirred at 100 °C for 16 h. The resulting white solid was collected by filtration, washed with EtOH (30 mL), and dried in vacuo to give 4-(pyridin-2-yl)-3,4-dihydrobenzo[4,5]imidazo[1,2-a][1,3,5]triazin-2-amine as a white solid (2.7 g, 89% yield). 1 H NMR (400 MHz, DMSO-d6): δ 8.56 (d, J = 4.4 Hz, 1H), 8.09 (s, 1H), 7.82 (t, J = 7.0 Hz, 1H), 7.38 - 7.35 (m, 1H), 7.25 - 7.22 (m, 2H), 6.95 - 6.88 (m, 2H), 6.83 - 6.77 (m, 2H), 6.44 (brs, 2H).

[0251] 2,2,5-Tris(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one (NIRA2):

[0252]

[0253] Under N2, piperidine (38.81 mg, 0.46 mmol) was added to a mixture of 4-(pyridin-2-yl)-3,4-dihydrobenzo[4,5]imidazo[1,2-a][1,3,5]triazin-2-amine (200 mg, 0.76 mmol) and 1,2-bis(2-pyridyl)ethane-1,2-dione (210 mg, 0.99 mmol) in EtOH (2 mL) and DMSO (4 mL). The mixture was stirred at 120 °C for 16 h. The mixture was quenched with water (40 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by trituration with EtOAc (30 mL), filtered, and the yellow solid was dried in vacuo to afford 2,2,5-tris(pyridin-2-yl)-1,2-dihydrobenzo[4,5]imidazo[1,2-a]imidazo[2,1-d][1,3,5]triazin-3(5H)-one as a yellow solid (80 mg, 23% yield). 1 1H NMR (400 MHz, DMSO-d6): δ 10.43 (brs, 1H), 8.59 (d, J = 4.0 Hz, 1H), 8.49 (d, J = 4.0 Hz, 1H), 8.38 (d, J = 4.0 Hz, 1H), 7.88 - 7.83 (m, 4H), 7.66 (s, 1H), 7.42 - 7.31 (m, 7H), 7.00 - 6.95 (m, 2H).

[0254] Immunoblotting. Cells were lysed in RIPA buffer supplemented with 1% SDS, protease (Sigma P8340) and phosphatase inhibitors (Roche 04906845001). Lysates were separated on 12%, 15% or 4 - 20% SDS-PAGE and transferred to PVDF membranes (Bio-Rad TurboBlot). Membranes were blocked in 5% BSA in TBS-T (0.05%) and incubated overnight with primary antibodies. Blots were washed, probed with appropriate secondary antibodies and processed with ECL (film or Bio-Rad chemiluminescence system) or imaged on a SpectraMax i3 platform (Molecular Devices) with a ScanLater module.

[0255] Production and transduction of retrovirus. pBabe puro HRasG12V The plasmid was from Addgene (#9051). HRas was prepared by restoring G12V to G using site-directed mutagenesis. WT The plasmid was verified by Sanger sequencing (CCHMC DNA Core). Retroviral supernatant was generated by transfecting HEK293T cells with pCL-Eco and pBabe-puro plasmids at a 1:1 ratio using the calcium phosphate method (Trono Lab). Supernatants were collected and filtered 24 and 48 hours after transfection. NIH / 3T3 cells were transduced overnight in the presence of polybrene (10 μg / mL) and selected with puromycin (3 μg / mL). Production and manipulation of retroviruses were performed in a BSL-2 facility.

[0256] Anchorage-independent growth assay. The bottom agar layer was prepared by mixing 2X complete DMEM with 1% agar (BD) in a 12-well plate to a final concentration of 0.5% and allowing it to solidify. For the top agarose layer of each well, 2,500 cells in 2X complete DMEM were resuspended in 0.6% low melting point agarose (IBI Scientific) and layered on top of the bottom agar layer. The next day, 100 μL of complete medium containing the test compound was overlaid on the upper layer to prevent drying. The medium was renewed twice a week. After 21 days, colonies were stained with 0.1% iodonitrotetrazolium violet (Sigma-Aldrich), imaged using an EVOS microscope (Life Technologies), and counted.

[0257] Sphere formation assay. Spheres were formed by the hanging drop method or using ultra-low attachment (ULA) plates (Corning). For the hanging drop assay, cells were trypsinized and resuspended at a concentration of 25,000 cells / mL in complete medium containing vehicle (DMSO) or the indicated concentration of IODVA1, and plated as 25 μL droplets on the inverted lid of a 10-cm culture dish. The dish was filled with 7 mL of PBS, the lid was placed on, and the cells were incubated for 3 - 5 days. For mechanical testing of sphere compaction, spheres were first imaged on an EVOS microscope, pipetted up and down 7 - 9 times, and then imaged again to assess sphere disruption. For ULA-based sphere formation, 5,000 cells were resuspended in 500 μL of complete medium containing vehicle (DMSO) or the indicated concentration of IODVA1 and plated in a 24-well ULA plate (Corning). Sphere formation was monitored daily and imaged using an EVOS microscope (Life Technologies). To assess attachment-independent proliferation, the contents of the wells were transferred to Eppendorf tubes, centrifuged at 100 x g for 5 minutes, dissociated into single cell suspensions by treatment with trituration and Accutase (Invitrogen) at room temperature, and counted using trypan blue exclusion.

[0258] Active GTPase binding assay: The levels of active Ras, Rac, Cdc42, and RhoA were determined using the Active Ras Pull-Down and Detection Kit, Active Rac Pull-Down and Detection Kit (Thermo Scientific), and RhoA / Rac1 / Cdc4 Pull-Down Activation Assay Combo Biochem Kit (Cytoskeleton). Cells were cultured and treated as indicated and lysed in the buffer provided by the manufacturer. The clarified whole cell lysate was incubated with recombinant GST-Raf1-RBD (for active Ras) (amino acids 1 - 149) and glutathione beads (both provided by the manufacturer), GST-Rhotekin-RBD (for active RhoA), GST-PAK-GBD (for active Rac and Cdc42) at 4 °C for 1 hour, washed, and the resulting complexes were eluted from the resin by boiling in 2X SDS sample buffer. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and the levels of active GTPases relative to the input lysate were determined by immunoblot analysis using anti-Ras, anti-Rac, anti-Cdc42, or anti-RhoA antibodies provided by the manufacturer.

[0259] Kinase assay: Reaction Biology ( http: / / www.reactionbiology.com)The activity of 369 wild-type kinases in the presence of a single dose of IODVA1 was measured using a HotSPot miniaturized radioisotope filter binding assay platform (Anastassiadis et al., 2011). Briefly, for each reaction, the kinase and substrate were mixed in a buffer containing 20 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij 35, 0.02 mg / ml BSA, 0.1 mM Na3VO4, 2 mM DTT, and 1% DMSO. Then IODVA1 was added to each reaction mixture to a final concentration of 0.5 μM. After incubation for 20 minutes at room temperature, the reaction was initiated by adding ATP (Sigma-Aldrich) and [γ- 32 P]-ATP (PerkinElmer, specific activity 10 Ci / L). The reaction was incubated for 2 hours at room temperature and then spotted onto P81 ion-exchange cellulose chromatography paper (Whatman). The filter paper was washed in 0.75% phosphoric acid to remove excess ATP. The percentage of remaining kinase activity for each kinase / IODVA1 pair relative to the vehicle (DMSO) kinase reaction was calculated. Each kinase inhibition assay was performed in duplicate and averaged. The data were processed and analyzed in Excel.

[0260] Immunofluorescence analysis of the actin cytoskeleton: MDA-MB-231 cells were seeded at a density of 2 x 10 4 cells per chamber in 8-well glass slides with or without EGF (5 ng / ml) for 10 minutes. After treatment with 1 μM IODVA1, the cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, and then stained with phalloidin conjugated to Alexa Fluor 568 (1:100) and DAPI and ProLong Gold (both from Life Technologies, Thermo-Fisher). The staining was visualized using a Nikon A1R confocal microscope.

[0261] Immunofluorescence and microscopy: To evaluate the initiation and maturation of lamellipodia, assays were performed in two ways. 1) MDA-MB-231 cells were plated on fibronectin-coated coverslips in serum-free medium for 4 hours and then incubated with IODVA1 (0 - 3 μM) in serum-free medium for 1 hour. The cells were then stimulated with EGF (50 ng / mL) for 30 minutes to induce lamellipodia formation. The cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton-X 100 and stained with phalloidin Alexa Fluor 568 or 594, and mounted in ProLong Gold DAPI (Life Technologies). 2) MDA-MB-231 cells were seeded at a density of 2 x 10 4 cells per chamber in 8-chamber slides with or without EGF (5 ng / ml) for 10 minutes. After treatment with 1 μM IODVA1, the cells were fixed and processed as in 1). The stained cells were observed using a Nikon A1R confocal microscope. Ten random fields were imaged and analyzed for lamellipodia formation. To evaluate the formation of circular dorsal ruffles, NIH-3T3 cells were treated as described by (Steffen et al., 2013). Briefly, cells (4 x 10 4 ) were plated on fibronectin-coated coverslips, serum-starved for 4 hours, and treated with IODVA1 (0–3 μM) in serum-free medium for 1 hour. Wrinkle induction was performed with PDGF BB (50 ng / mL, Peprotech) for 10 minutes. The cells were fixed in 4% paraformaldehyde and immunofluorescence microscopy was performed as described above. Approximately 150 cells were counted per treatment group per experiment. To evaluate stress fibers, cells were plated on fibronectin-coated coverslips in serum-free medium for 4 hours, incubated with IODVA1 (0–3 μM) for 1 hour, fixed, and treated with fluorescent phalloidin as before.

[0262] For cell spreading, MDA-MB-231, MCF7, and T47D cells were seeded on fibronectin-coated coverslips in serum-free medium. After 10 minutes, IODVA1 (0 - 3 μM) was added to the serum-free medium. After 30 minutes (total 40 minutes), the cells were fixed in 4% paraformaldehyde and imaged using bright-field microscopy (EVOS, Life Technologies). Six random fields were imaged at 20x magnification and the cell area of individual cells was quantified to evaluate the degree of spreading (ImageJ). More than 300 cells were counted per treatment group per experiment.

[0263] In vivo analysis using the MDA-MB-231 xenograft mouse model: For xenograft studies, 1 x 106 MDA-MB-231 cells were suspended in PBS and injected into each inguinal mammary fat pad of 10-week-old virgin female athymic nude mice. Tumors were measured weekly with a digital caliper, and volume was calculated as [(π / 6)) x L x W 2 (Euhus et al., 1986; Tomayko and Reynolds, 1989). Treatment began eight weeks after injection, when tumors reached 200 mm 3 . Mice received an intraperitoneal injection of 250 μL of diluent (5% DMSO, in PBS) or 1 mM compound IODVA1 three times a week for 4 weeks, with an average drug dose of 3.5 mg / kg. At necropsy, mice were weighed, and tumors were excised, measured, weighed, fixed in 4% paraformaldehyde, and embedded in paraffin. Peripheral blood was collected by cardiac puncture and analyzed for a complete blood count with a Hemavet (Drew Scientific, Miami Lakes, FL, USA). The use and handling of mice were conducted under the approval of the Cincinnati Children's Institutional Animal Care and Use Committee. All mice were housed in specific pathogen-free conditions with free access to food and water.

[0264] Histology: Tissues were fixed in 4% paraformaldehyde and then paraffin-embedded tissues were sectioned at 5 μm. Tissues were stained with hematoxylin and eosin (H&E) or by immunofluorescence staining. Tissue sections were subjected to citrate antigen retrieval, blocked with 10% normal goat serum, and incubated with Ki67 antibody (1:100, Abcam) or cleaved caspase 3 (Asp175, 1:100, Cell Signaling), and then incubated with goat anti-rabbit: Alexa568 (1:500, Abcam). Tissues were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and coverslipped with VectaShield HardSet (Vector Labs). Images were acquired by confocal microscopy (Nikon) and immunofluorescence analysis was performed using Image J.

[0265] In vivo analysis of the H2122 xenograft mouse model. The human lung cancer cell line NCI-H2122 (ATCC) carrying a bi-allelic KRASG12C mutation was cultured in RPMI-1640 supplemented with 10% FBS. For injection, NCI-H2122 cells were trypsinized and suspended at a concentration of 2 x 10 7 cells / mL in 50% Matrigel (Corning). 100 μl of the cell suspension was subcutaneously injected into 6-week-old NOD.Cg-Prkdc scid Il2rg tm1WjlIn / SzJ(NSG) mice. Starting from 10 days after xenotransplantation, the mice were given intraperitoneal injections every other day for 14 days (a total of 7 injections). A 200 mM IODVA1 stock solution in DMSO was freshly diluted 1:19 in DMSO and then 1:9 in PBS to achieve a final concentration of 1 mM in 10% DMSO. The mice were injected with 250 μl of vehicle (10% DMSO) or IODVA1 (1 mM). Tumor size was measured using a digital caliper, and tumor volume was estimated using the formula (length x width 2 ) / 2. For immunostaining, paraffin-embedded sections were stained with Ki-67 antibody (clone SP6, ThermoFisher) at a dilution of 1:1,000. DAB-stained slides were counterstained with anti-nuclear red dye. To quantify Ki-67 positive cells, random fields were imaged at 200x magnification and quantified using Image J software. At least 20 random fields were used for quantification. Scale bar = 100 μm.

[0266] Note: Mitotic cells were easily visible in vehicle-treated mice. IODVA1 increased intratumoral fibrosis. Both vehicle and IODVA1 tumors had central necrosis, which is typical of subcutaneous tumors of this size.

[0267] Inhibition of Rac activity

[0268] Rac GTPases (Rac1, Rac2, Rac3, and RhoG) are tightly regulated signaling switches that mediate inputs from various receptors and oncogenes to regulate growth, apoptosis, cell-cell, and cell-matrix interactions in response to growth factors such as EGF, PDGF, and HGF. Regulation of the actin cytoskeleton, which plays a key role in cell shape, polarity, division, migration, and metastasis, is a major function of Rac, as it promotes membrane ruffling and the formation of lamellipodia and circular dorsal ruffles (Etienne-Manneville, 2002; Jaffe and Hall, 2005; Bustelo, 2018; Ridley and Hall, 2015; Ridley, 1992; Steffen, 2013). Rac also controls cell cycle progression and cell survival, integrin-mediated adhesion, and is required for Ras transformation (Coleman, 2004; Kiosses, 2001; Sundaresan, 1996; Mack, 2011; Qiu, 1995). In addition, Rac plays a key role in most aggressive leukemias (Thomas, 2007; Somervaille, 2006; Wei, 2008; Sengupta, 2010; Skorski, 1998; Mizukawa, 2011; Bassermann, 2002; Nieborowska-Skorska, 2012). Thus, Rac is associated with pro-tumorigenic functions and is involved in cancer development. In addition, Rac is characterized by resistance to chemotherapy, radiotherapy, and targeted therapies and is associated with the persistence of leukemia stem cells (Jaffe and Hall, 2005; Loirand, 2010; Mulloy, 2010; Newey, 2005; Sahai, 2002; Vigil, 2010; Porter, 2016; Zandvakili, 2017; Cardama, 2018).

[0269] Reducing Rac activity, especially in cancer cells, is desirable and an active area of research. However, despite many efforts, no small molecule inhibitor of Rac signaling is in clinical use. Rac activity is regulated by a complex and choreographed set of proteins consisting of guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine dissociation inhibitors (GDIs). RacGEFs activate Rac by exchanging the bound GDP for GTP to initiate signaling, while GAPs inactivate Rac by increasing the rate of GTP hydrolysis to block signaling. GDIs extract Rac from the membrane, thereby preventing it from signaling. When activated, Rac binds to and activates downstream effectors such as p21-activated kinases (PAK1 / 2 / 3), which in turn activate prosurvival pathways and actin regulatory proteins. Rac regulators and effectors themselves are tightly regulated. For example, Vav proteins (Vav1, Vav2, and Vav3) are multi-domain tyrosine phosphorylation-dependent RacGEFs. Phosphorylation of specific tyrosine residues releases N-terminal and C-terminal autoinhibitory mechanisms, thereby allowing Rac access to the Dbl homology (DH) domain required for the GTP exchange reaction. Thus, finding small molecule inhibitors of Rac itself or its activators, such as Vav proteins, would provide an effective strategy for treating malignancies with aberrant Rac signaling.

[0270] In this study, the applicant has revealed the mechanism of action (MoA) of IODVA1, a 2-guanidinobenzimidazole derivative that was identified as the active ingredient in NSC124205. Preliminary characterization of IODVA1 indicates that it is not a kinase inhibitor, but it can prevent the formation of lamellipodia and circular dorsal ruffles at low concentrations and within minutes of cell exposure. It also reduces cell-cell and cell-extracellular matrix interactions and reduces the growth of Ras-driven tumors. These properties and the specificity of IODVA1 for cells expressing oncogenes suggest its targeting of Rac activation. The applicant used the chimeric BCR-ABL1 oncoprotein B cell acute lymphoblastic leukemia (Ph +In vitro and in vivo leukemia models of BCR-ABL1 B-ALL were used to study the MoA of IODVA1. For multiple reasons, the BCR-ABL1 B-ALL model is well-suited for this work. First, BCR-ABL B-ALL is a single-driver gene model. The expression of BCR-ABL with constitutive kinase activity is sufficient to confer a growth advantage to leukemia cells. When expressed, BCR-ABL1 activates multiple pathways, including the Ras-mitogen-activated protein kinase (MAPK) pathway that leads to abnormal cell proliferation, the Janus-activated kinase (JAK)-STAT pathway that results in impaired transcriptional activity, and the phosphoinositide 3-kinase (PI-3K) / AKT pathway that leads to extended survival (Cilloni, 2012). In addition, the expression of p190- or p210-BCR-ABL activates the Rac signaling pathway to regulate leukemogenesis (Skorski, 1998; Thomas, 2008; Harnois, 2003; Sahay, 2008) and deletion of Rac2 or the combination of Rac1 and Rac2 impairs p210-BCR-ABL expression-induced myeloid leukemogenesis in the hematopoietic stem and progenitor cell compartment (Thomas, 2007; Sengupta, 2010). Second, the seemingly complex pathways activated by BCR-ABL all rely on the dysregulated kinase activity of BCR-ABL (Lugo, 1990), and ABL1-tyrosine kinase inhibitors (ABL1-TKIs), such as imatinib, are used as first-line therapies. Thus, imatinib can be used as a positive control to evaluate the efficacy of IODVA1. Third, despite their great success in treating B-ALL clinically, the emergence of TKI-dependent and -independent resistance mechanisms limits their efficacy (Arrigoni, 2018; Hamilton, 2012). Thus, there is an unmet need for novel therapies for TKI-resistant leukemia patients and for therapies to prevent the persistence of leukemia cells.

[0271] Here, the applicant shows that IODVA1 tightly binds and inhibits Vav3, thus leading to the inactivation of Rac and its downstream signaling and specifically inducing apoptosis of cells expressing BCR-ABL in vitro and in vivo. In addition, the applicant shows that IODVA1 prolongs the survival of a TKI-resistant mouse model and reduces its leukemia burden for a long time after treatment cessation. The applicant also shows that IODVA1 effectively reduces the proliferation and survival of recurrent and de novo primary patient-derived cells. It is believed that IODVA1 is the first RhoGEF inhibitor with in vivo activity against cancer xenograft mouse models. The applicant's findings have direct implications for overcoming TKI resistance in the clinic and treating cancers targeted by Vav3, including Ras-driven cancers.

[0272] Results

[0273] IODVA1 specifically targets BCR-ABL B-ALL cells in vitro. To confirm that IODVA1 is specific for cells expressing oncogenes, we tested its efficacy on the proliferation and survival of CD34 + human peripheral blood mononuclear cells transduced with the retroviral bicistronic p190-BCR-ABL or Mieg3 empty vector (Williams, 2000). As expected, the expression of BCR-ABL increased cell proliferation ( Figure 10 A). Treatment with IODVA1 (IO1, 1 μM) decreased the proliferation of BCR-ABL-transformed cells, while the proliferation of cells transduced with the empty vector Mieg3 was unaffected. We then evaluated the viability of p190-BCR-ABL-transformed CD34 + cells in the presence of IODVA1 by trypan blue exclusion. The viability of cells expressing p190-BCR-ABL decreased to 60 ± 16% (SEM, N = 3) at 1 μM on day 5 and to 1 ± 0.2% (SEM, N = 3) at 3 μM on day 3 in a dose-dependent manner ( Figure 10 B). The viability of cells expressing the empty vector was unaffected by IODVA1. IODVA1 irreversibly inhibited the viability of Ba / F3 cells expressing p190 and p210-BCR-ABL1 but not that of Ba / F3 cells expressing the empty vector (Mieg3), with a half-maximal effective concentration (EC50) of 380 nM, an EC50 of 680 nM for Nalm-1 cells, and inhibited the colony-forming ability of BCR-ABL1-transformed Ba / F3 cells in soft agar ( Figure 11 A-11D). Collectively, these results indicate that IODVA1 specifically targets the proliferation and survival of BCR-ABL1-transformed cells and, consistent with our previous report, is more specific for cells expressing oncogenes (Gasilina et al., 2020).

[0274] IODVA1 prevents leukemia-related death and significantly reduces the leukemia burden in a BCR-ABL-induced murine leukemia model. To test whether the efficacy of IODVA1 on cells could be recapitulated in vivo, the applicant explored its efficacy on a p190-BCR-ABL-induced murine B-ALL model and compared it with imatinib, an ABL1-TKI with a well-characterized MoA in the Ph + B-ALL mouse model and is clinically used as a Ph +The first-line therapies for induced malignancies) were compared. C57Bl / 6 mice were used as donors and / or recipients for the transduction / transplantation model. Mouse low-density bone marrow (LDBM) cells were transduced with an MSCV-driven bicistronic retroviral vector (MSCV-IRES-EGFP) encoding p190-BCR-ABL. The transduced LDBM cells (1×10 6 ) were intravenously transplanted into lethally irradiated C57Bl / 6 mice. The mice were bled after 23 days, and GFP + cells were analyzed by flow cytometry. By day 28, all mice had developed leukemia. The leukemic mice were divided into 5 groups (7 mice per group) and administered PBS control vehicle, 0.25 or 0.5 mM IODVA1, 0.5 mM imatinib, or a combination of 0.25 mM IODVA1 + 0.5 mM imatinib. The vehicle control group had the same amount of DMSO (0.1%) as the other groups. The drugs were administered subcutaneously in osmotic pumps for 4 weeks of sustained release. Mouse survival plots showed that while the control group died within 7 to 10 days after administration of PBS vehicle, the low IODVA1 dose (0.25 mM) increased survival by an average of 10 days. Mice treated with imatinib or 0.5 mM IODVA1 or the combination remained alive throughout the 4-week duration of treatment ( Figure 10 C). Significantly, IODVA1 reduced the residual p190-BCR-ABL-expressing leukemic progenitor B cells (EGFP + / B220 + ) from the peripheral blood (PB) of treated mice ( Figure 11 E).

[0275] IODVA1 eradicated the leukemic dissemination activity evaluated by serial transplantation. Despite significant clinical success, imatinib and more generally TKIs cannot eliminate leukemic stem / progenitor cells in the bone marrow (BM), which can lead to residual disease, the emergence of resistance mechanisms, and ultimately relapse (Milojkovic, 2009; Bixby, 2009). To determine whether IODVA1 eradicates progenitor B cells with the ability to proliferate tumors (a functional surrogate for minimal residual disease that may lead to leukemia relapse), BM cells from Figure 10 the vehicle control and treated mice of 6 C (excluding mice treated with 0.25 mM IODVA1) were transplanted into secondary lethally irradiated C57BL / 6 mice in a limiting dilution series at cell doses of 1x10 6 , 0.3x10 6 , and 0.1x10 6Kaplan-Meier survival plots of single-cell diluted transplantation showed that administration of IODVA1 alone or in combination with imatinib led to survival beyond the 70-day endpoint analysis in p190-BCR-ABL chimeric mice ( Figure 10 D). Mice transplanted with BM cells from primary recipient mice treated with imatinib alone died on day 40 post-transplantation. Analysis of leukemia progenitor cells (EGFP + / B220 + ) in the peripheral blood of secondary transplanted mice at 5 weeks post-transplantation ( Figure 11 F) showed that IODVA1 was superior to imatinib in eradicating leukemia cell burden. Poisson distribution analysis of lower cell dose grafts ( Figure 11 G-11J) showed that the depletion of tumor-propagating activity in grafts from leukemia mice treated with IODVA1- or IODVA1 + imatinib was >10-fold compared to leukemia mice treated with imatinib alone.

[0276] IODVA1 eradicates TKI-resistant BCR-ABL B-ALL. Since IODVA1 has no inhibitory activity against major wild-type kinases including ABL1 and SRC-like kinases, the anti-proliferative activity of IODVA1 against BCR-ABL B-ALL models in vitro and in vivo and its ability to eradicate residual disease cannot be explained by ABL1 inhibition. To further test this idea, we evaluated the ability of IODVA1 to increase the survival of mice carrying p210-BCR-ABL (T315I). We chose this ABL1 mutant because it is one of the most common mutations in chronic myeloid leukemia (CML) patients treated with imatinib (Gorre, 2001; Azam, 2003; Jabbour, 2006; Nicolini, 2006; Jabbour, 2008). Mice were treated for 4 weeks with two rounds of pumps containing vehicle control, imatinib, or IODVA1. At the end of the 4-week treatment, surviving mice were caged without any additional treatment. As expected, p210(T315I) mice were unresponsive to TKI as all imatinib-treated mice died on day 22 before the end of treatment ( Figure 12 A). 80% of IODVA1-treated mice survived until day 65, day 37 after the last treatment. 60% of IODVA1-treated mice survived until day 80, day 52 after the last treatment ( Figure 12 A). Analysis of EGFP + / B220 +The counting of leukemia progenitor cells showed that IODVA1 significantly reduced the level of leukemia progenitor cells by 24% at the second week, 84% at the 5th week, and 91% at the 10th week( Figure 12 B).

[0277] To evaluate the signaling pathways affected by IODVA1, EGFP + / B220 + LDBM cells were isolated from p210(T315I) mice treated for two weeks, stained with phosphorylation antibodies against the pro-proliferative Rac-dependent effectors JNK, PAK, 4EBP, and S6, and the Rac-independent effectors ERK1 / 2, STAT3, STAT5, p38, and AKT, and analyzed by flow cytometry. IODVA1 caused a significant decrease in pJNK by 55% (p = 0.0029), pPAK by 56% (p = 0.0016), p4EBP by 20.3% (p = 0.037), and pS6 by 17.8% (p = 0.0012)( Figure 12 C). The phosphorylation levels of p38, ERK, STAT3, STAT5, and AKT were not affected by IODVA1. Interestingly, imatinib had the opposite effect, reducing the levels of pERK, pSTAT3, and pAKT, but not affecting the phosphorylation levels of Rac-dependent effectors. Taken together, IODVA1 not only overcame TKI resistance but also eliminated TKI-resistant leukemia stem / progenitor cells by acting on the imatinib-independent growth signaling pathway involving Rac effectors.

[0278] IODVA1 reduces Rac activity and downstream signaling. It has been shown that IODVA1 blocked the formation of F-actin superstructures, such as lamellipodia and circular dorsal ruffles, within minutes of cell cultivation( Figure 5 A-5B and Gasilina et al., 2020), and we focused on Rac. Rac is a major regulator of lamellipodia formation and JNK and TORC1 activities (Minden, 1995; Saci, 2011), and is required for the formation of circular dorsal ruffles and is also activated downstream of BCR-ABL. Therefore, we tested whether IODVA1 inhibited Rac activation and measured the levels of Rac-GTP during treatment with PAK-GBD (GTPase-binding domain). p190-BCR-ABL-transformed Ba / F3 cells were treated with IODVA1 (3 μM, Figure 13A) After co-culture for 10 - 15 minutes, the active Rac level decreased by 70%. Interestingly, this 10-minute time point was consistent with the reduction of lamellipodia formation in IODVA1-driven MDA-MB-231 breast cancer cells ( Figure 5 C). IODVA1 is specific for Rac (IC50 = 1 μM), has a poorer effect on Cdc42, and is ineffective against RhoA ( Figure 13 G). Interestingly, the rapid action and specificity of IODVA1 for Rac were consistent with MDA-MB-231 breast cancer cells.

[0279] To test whether the reduction of Rac activation was translated into a reduction of downstream signaling in vitro, as observed in xenograft-derived TKI-resistant cells ( Figure 12 C), we analyzed vehicle- and IODVA1-treated Ba / F3 cells expressing p190-BCR-ABL or empty vector by phospho-flow cytometry. The expression of BCR-ABL1 increased the phosphorylation levels of JNK, S6, 4EBP, PAK, and AKT by more than 2.5-fold ( Figure 13 B). IODVA1 decreased the phosphorylation level of JNK by 1.8 (p = 0.015)-fold, S6 by 1.5 (p = 0.05)-fold, 4EBP by 3.0 (p = 0.009)-fold, and PAK by 6.1 (p = 0.004)-fold, respectively. Importantly, the reduction of effector phosphorylation levels induced by IODVA1 was specific to cells expressing BCR-ABL1, rather than cells expressing empty vector. Regardless of the oncogene BCR-ABL status, IODVA1 did not affect the phosphorylation level of AKT ( Figure 13 B). The decrease in JNK, S6, and 4EBP activities in IODVA1-treated Ba / F3 cells reflected the decrease observed in LDBM cells from IODVA1-treated p210-T315I mice in the pharmacodynamic study ( Figure 12 C). Collectively, our in vitro and in vivo data were consistent with IODVA1 targeting Rac activation and its downstream signaling.

[0280] Significantly, IODVA1 decreased pro-survival PAK downstream of Rac and reduced the inhibitory phosphorylation of pro-apoptotic BADSer136 within minutes of cell exposure ( Figure 13 F). The reduction of PAK and BAD phosphorylation indicated that IODVA1 promoted a decrease in survival rate and induction of apoptosis. To further validate this hypothesis, we analyzed murine p190-BCR-ABL leukemia progenitor cells (EGFP + / B220dim ) cell cycle and then performed in vitro BrdU incorporation and flow cytometry analysis ( Figure 13 C). IODVA1 did not affect the G0+G1 phase, and it significantly affected the distribution of the G2+M, S, and apoptotic phases. It increased the percentage of cells in the G2+M phase from 7±1% (SD, N = 3) in the presence of vehicle control to 22±6.6, 32±3, and 36±7% (SD, N = 3) at 1, 3, and 10 μM, respectively. At 1 μM, it decreased the S phase by 8-fold (p = 0.0002) and increased apoptosis by at least 5.3-fold (p = 0.007). Thus, IODVA1 induced G2 / M arrest.

[0281] Rac-deficient cells did not respond to IODVA1. To confirm that IODVA1 targets the Rac-dependent pathway, we evaluated its effect in a Rac2-null background. Rac1Δ / Δ+Rac2 - / - murine leukemia cells exhibited a severe reduction in Rac1 expression and lacked Rac2 ( Figure 13 H). The clonogenic ability of Rac1Δ / Δ+Rac2 - / - expressing p190-BCR-ABL or wild-type murine leukemia cells (Thomas, 2008) was tested in the presence of IODVA1 ( Figure 13 D). Rac1Δ / Δ+Rac2 - / - leukemia cells formed 3.8-fold fewer colonies than wild-type leukemia cells (p = 0.0003). IODVA1 did not alter the number of colonies formed by Rac1Δ / Δ+Rac2 - / - cells, indicating that these cells were insensitive to IODVA1. Interestingly, Rac1Δ / Δ+Rac2 - / - leukemia cells treated with vehicle or IODVA1 formed 2.4-fold more colonies than those of wild-type cells treated with IODVA1 (p = 0.0091). Combining the biochemical data, these data support the view that IODVA1 targets Rac activity and thus inhibits its downstream prosurvival signals and induces G2 / M arrest associated with apoptosis.

[0282] IODVA1 is an inhibitor of RacGEF Vav3. Rac activity and signaling are regulated by GAP, GDI, and RacGEF. We hypothesized that the decrease in Rac activity might be caused by IODVA1 targeting a Rac regulator. Using biochemical analysis, we showed that IODVA1 did not stimulate the activity of the Rac negative regulators p50GAP and RhoGDI1 (Figure 13 I-13K). We next turn to the GEF and assume that IODVA1 inhibits a Rac-specific GEF, leading to its inactivation. Although several Rac GEFs have been associated with leukemogenesis (Biswas, 2019; Chatterjee, 2018; Martin, 2013; Lyons, 2010; Reuther, 2001; Rouard, 1999; Bourgoin, 1995), Vav3 has been shown to play an important role in leukemogenesis (Chang et al., 2012). Therefore, we focused on Vav3 and tested whether IODVA1 inhibits the binding of Vav3 to Rac. Ba / F3 cells expressing p190-BCR-ABL or the Mieg3 empty vector were incubated with IODVA1 (3 μM) or vehicle control for 30 minutes and then subjected to GST-Rac pull-down. The pulled-down protein complexes were separated on SDS-PAGE and immunoblotted and quantified against pVav3 and Vav3 ( Figure 14 A and 14E). In Ba / F3 cells expressing the empty vector treated with IODVA1 or vehicle control, there was no significant change in Vav3 or pVav3 bound to Rac ( Figure 14 A, lanes 1 and 2), indicating that IODVA1 does not affect the binding of Vav3 to Rac in cells expressing the empty vector. Strong pVav3 and Vav3 bands appeared in cells expressing the p190-BCR-ABL oncogene and treated with vehicle ( Figure 14 A, lane 3), suggesting a strong binding between active Vav3 and Rac. Specifically, a 5-fold increase in the intensity of pVav3 in Ba / F3 leukemia cells was consistent with a similar observation that BCR-ABL increases Vav3 activation (Chang, 2012). The intensity of this band was reduced 8-fold in cells treated with IODVA1 ( Figure 14 A, lane 4), indicating that IODVA1 inhibits the binding of Vav3 to Rac in BCR-ABL-expressing cells. This inhibition may be due to an overall decrease in the level of pVav3 in IODVA1-treated cells ( Figure 14 A, input pVav3 band, lanes 3 and 4). Collectively, our data indicate that IODVA1 interferes with the activation of Vav3 and its binding to Rac, thereby preventing the activation of the latter.

[0283] IODVA1 binds to Vav3. Previous observations have shown that IODVA1 binds to Vav3. To verify this hypothesis, we measured its binding affinity (Kd) for recombinant Vav3 and Rac1 using microscale thermophoresis (MST); the catalytic domain (DH / PH) of RhoGEF LARG served as a negative control. We used LARG for two reasons. First, we reasoned that if Rho activation was not affected by IODVA1 ( Figure 13 G), we should not detect any binding between LARG and IODVA1. Second, like Vav3, LARG contains a DH / PH domain responsible for exchange activity and thus any non-specific binding to this domain should be detected. The MST signal for Vav3 saturated at 10 μM IODVA1 and above ( Figure 14 B). The MST signals for Rac1 and LARG did not show saturation at the highest IODVA1 concentration tested. Fitting of the MST titration data indicated that one molecule of IODVA1 bound to Vav3 with a K d of 512 nM. The best estimates of the K d for Rac and LARG were 35.5 and 7.7 μM, respectively. Thus, IODVA1 binds tightly and specifically to Vav3.

[0284] Vav3-deficient leukemia cells are unresponsive to IODVA1 in vitro and in vivo. To further validate Vav3 as a target of IODVA1, we investigated the effect of IODVA1 on the leukemia cells of the Vav3-KO (Vav3 - / - ) mice we had previously published (Thomas EK et al., 2007; Thomas EK et al., 2008; Bourgoin S. et al., 1995). We reasoned that if IODVA1 targets Vav3, then Vav3 - / - cells should be insensitive to its action. In the presence of IODVA1, wild-type or Vav3 + / B220 + ) murine BM leukemia cells expressing p190-BCR-ABL (EGFP - / - ) were tested in a colony formation assay ( Figure 14 C). In the presence of 1 and 3 μM IODVA1, the number of colonies formed by leukemia cells expressing Vav3 decreased by an average of 3- and 7-fold, respectively. On the other hand, Vav3 - / - cells formed a similar number of colonies when grown in the presence of vehicle control or IODVA1, indicating that these cells had lost sensitivity to our drug. Interestingly, by Vav3 - / -There was no statistically significant difference in the number of colonies formed by leukemic cells and wild-type leukemic cells treated with IODVA1 (p = 0.13). Similarly, cell cycle analysis showed that Vav3-expressing p190-BCR-ABL - / - cells were not affected by IODVA1( Figure 14 D). Collectively, our data indicate that while wild-type leukemic cells respond to IODVA1, Vav3 - / - leukemic cells do not respond and mimic wild-type leukemic cells treated with IODVA1.

[0285] Vav3 rescues IODVA1 sensitivity. Next, we reasoned that if IODVA1 targets Vav3, then rescuing Vav3 by expressing exogenous Vav3 - / - leukemic cells should render these cells sensitive to IODVA1 again. We expressed full-length Vav3 or the dominant-active ΔCH mutant in Vav3 - / - p190-BCR-ABL1-transformed murine myeloid leukemia cells( Figure 15 A), and analyzed the changes in the cell cycle 18 hours after treatment with vehicle control or IODVA1( Figure 15 B). Expression of full-length Vav3 but not the empty vector rendered Vav3 - / - BM leukemic cells sensitive to IODVA1 again, as shown by a 5.8-fold increase in apoptosis and a 15% decrease in cells in the S phase at 10 μM (p = 0.0005). Expression of the ΔCH mutant did not render Vav3 - / - leukemic cells sensitive to IODVA1 even at the highest concentration.

[0286] To test whether the results of the previous rescue experiments hold in a proliferation assay, we performed a colony formation assay on Vav3-expressing full-length and ΔCH mutant Vav3 - / - leukemic cells in the presence of IODVA1 (1, 5, and 10 μM) or vehicle control( Figure 15 C). Reintroduction of full-length or ΔCH Vav3 led to a number of colonies similar to that of wild-type BM leukemic cells (200 and 183 vs. 188, respectively), which was 3-fold higher than that of Vav3 - / - leukemic cells expressing the empty vector. Importantly, Vav3-expressing Vav3 - / - leukemic cells responded to IODVA1 in a dose-dependent manner. At 10 μM IODVA1, the number of colonies produced by Vav3 - / - cells expressing full-length Vav3 decreased by one-third and became similar to that of Vav3 - / -The number of colonies generated was similar (75 colonies vs. 66 colonies). Interestingly, leukemic cells expressing ΔCH showed a reduced response to IODVA1.

[0287] Vav3 - / - Leukemia did not respond to IODVA1 in vivo. Next, we tested whether the lack of response of Vav3 - / - cells to IODVA1 holds true in vivo. We transplanted wild-type or Vav3 - / - LDBM cells transduced with p190-BCR-ABL retrovirus into lethally irradiated C57BL / 6 mice, waited for leukemia to develop, and treated the mice with vehicle control or IODVA1 by osmotic pump as before. Vav3 - / - leukemic mice did not respond to IODVA1, which supported the hypothesis that Vav3 is the in vivo target of IODVA1 ( Figure 14 F). Taken together, our data indicate that in cell and in vivo assays, Vav3-deficient leukemic progenitors do not respond to IODVA1, which is consistent with the view that Vav3 is the target of IODVA1 in vivo and in vitro. The persistence of leukemia in Vav3 - / - mice suggests that Vav3 - / - BCR-ABL leukemia has evolved an escape mechanism that relies on Rac-independent pathways such as AKT and STAT3 signaling pathways ( Figure 14 G and 14H).

[0288] IODVA1 targets Vav3 in MDA-MB-231 cells and xenograft tumors. Next, we tested whether IODVA1 is effective in another Vav3-dependent cancer model. We chose MDA-MB-231 triple-negative breast cancer cells because these cells express high endogenous levels of Vav3 (Chen, 2015) and because Vav3 is a recognized target in breast cancer (Chen, 2015; Lee, 2008; Aguilar, 2014; Citterio, 2012; Lorenzo-Martin. 2019). In addition, we have previously shown that these cells are sensitive to IODVA1 and that IODVA1 blocks tumor growth and induces apoptosis in MDA-MB-231 xenograft mice ( Figure 8 A-8C) and (Gasilina et al., 2020). First, we showed that reducing the Vav3 expression level by targeting shRNA severely reduced the proliferation of MDA-MB-231 cells. IODVA1 (0.1–1 μM) reduced the viability and proliferation of scrambled MDA-MB-231 in a dose-dependent manner, which had no effect on shVav3-expressing cells ( Figure 16A). Next, we incubated MDA-MB-231 cells with IODVA1 (3 μM) for 15 minutes and analyzed the level of phosphorylated Vav3 (pY173) by immunoblotting. Figure 16 B showed that IODVA1 treatment led to a significant decrease in the pY173 signal. Since phosphorylation of this Tyr173 indicates Vav3 activation, we conclude that IODVA1 inhibits Vav3 activity shortly after exposure.

[0289] To test whether IODVA1 inhibits Vav3 in vivo, we utilized the MDA-MB-231 xenograft tumors we generated ( Figure 8 A) and stained these tumors by immunohistochemistry for pVav3. Comparison of tumor sections stained for pVav3 with vehicle control and with IODVA1 treatment showed that pVav3 staining was significantly reduced in tumors treated with IODVA1 compared to those treated with vehicle control ( Figure 16 C). Thus, IODVA1 also inhibits Vav3 in vitro and in vivo in solid tumor models in a cell-independent manner.

[0290] IODVA1 reduces the viability of leukemia cells derived from patients. Consistent with our findings on peripheral CD34 + BCR-ABL cells ( Figure 10 A-10B), cells from a PDX model representing a pediatric Ph + B-ALL patient were found to be highly sensitive to IODVA1 in vitro ( Figure 17 ).

[0291] Cells from relapsed patient #2018-136 with Ph + (BCR-ABL1) were treated with the ABL-TKI dasatinib, the JAK-inhibitor ruxolitinib, a combination of dasatinib and ruxolitinib (das+rux), the CDK-inhibitor palbociclib, and IODVA1. Dasatinib (20 nM) reduced the proliferation of #2018-136 cells by 56%; ruxolitinib or palbociclib (1 μM) had no effect. The das+rux combination led to a 63% reduction in proliferation, likely due to the inhibitory effect of dasatinib. IODVA1 (0.5 μM) reduced the proliferation of these cells by 78%. When tested in a colony formation assay, IODVA1 (1 μM) reduced the number of colonies by 60% (p = 0.001) ( Figure 17 ).

[0292] For those with dual Ph +Cells from patient #2017-58 with (BCR-ABL1) and Ph-like (P2RY8-CRLF2) rearrangements were treated similarly. These cells responded significantly to dasatinib, ruxolitinib, and their combination. IODVA1 was less effective as it only reduced cell proliferation by 40% at 1 μM and had no effect at 0.2 μM ( Figure 17 B). Cells from patient #2017-129 with Ph + B-ALL (BCR-ABL1; T315I) at relapse after initial therapy were isolated and treated with vehicle control, dasatinib, ruxolitinib, (das+rux), and IODVA1. As expected, dasatinib, ruxolitinib, or the combination had no effect on the proliferation of CD19 + cells ( + Figure 17 C, left panel). In contrast, 1 μM but not 0.2 μM of IODVA1 reduced the CD19 Figure 17 + B-ALL cell count by 80%. In addition, we confirmed that IODVA1 did not have a toxic effect on cells of normal stroma ( Figure 17 C, right panel arrow). Thus, IODVA1 reduces the proliferation of primary Ph + B-ALL (BCR-ABL1) cells, including those expressing the TKI-resistant T315I mutant, which is consistent with our finding that Ph + B-ALL (BCR-ABL1) model cells express high levels of Vav3. The fact that #2017-58 cells did not respond to IODVA1 as well as the other two patient samples may be due to the presence of other genetic mutations (e.g., P2RY8-CRLF2) that promote cell growth independent of Vav3.

[0293] Our sample cohort also contained many cases of Ph-like disease with multiple genetic aberrations and some cases of MLL-rearranged B-ALL. Cells from these patients generally responded positively to IODVA1 ( Figure 18 ).

[0294] In summary, we have shown that pharmacological inhibition of Vav3 by IODVA1 is a potential treatment for Ph +An attractive therapeutic strategy for TKI-resistant BCR-ABL B-ALL. This strategy should benefit other malignancies targeting Vav3, such as other leukemias, breast cancers with poor prognosis, skin tumors, prostate cancers, gastric cancers, glioblastomas, or where Vav3 is highly expressed. Therefore, we expect IODVA1 to have broader therapeutic applications. In addition, IODVA1 constitutes a special tool for dissecting the Vav3 / Rac signaling axis. Broadly speaking, RhoGEF is a multi-domain protein regulated by auto-inhibition. Therefore, small molecules that stabilize the auto-inhibited conformation of RhoGEF and inhibit its activity can be developed into drugs for treating human cancers.

[0295] Plasmids, cell lines, and reagents: The plasmid set pMalX(A-E) for purifying the fixed-arm carrier fusion was a gift from Dr. Lars C. Pedersen (NIEHS). The pET28b-N9-MBP-mOrange plasmid was from Addgene (#29748). The molecular chaperone co-expression plasmid set was from TaKaRa (catalog number #3340). Primers were from Integrated DNA Technologies (IDT, Inc.). The NEBuilder tool was used for primer and construct design. Restriction enzymes, polymerases, cloning assembly kits, and competent cells were from New England Biolabs and Invitrogen.

[0296] MDA-MB-231 cells were maintained in IMEM (Invitrogen) supplemented with 10% FBS, 1% penicillin / streptomycin, and 1% amphotericin B. Ba / F3 cells were cultured in RPMI (GIBCO) supplemented with 10% FBS and IL-3 (10 ng / ml). HEK293T cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. All cells were cultured in a 5% CO2 humidified incubator at 37°C. Cell viability was evaluated by trypan blue exclusion assay as previously described. Cytokines were from Peprotech.

[0297] The following antibodies were used: GAPDH (#627408, GeneTex), pERK1 / 2 (#4370), pAKT (#9271 and #9018), c-Abl (#2862), Cdc42 (#2462), RhoA (#2117), pPAK1 / 2 (#2601S), pS6 (#4851S), PAK1 (#2602S), pBAD (#4366) and BAD (#9292), anti-mouse HRP (#7076), anti-rabbit HRP (#7074) from Cell Signaling Technologies, pVav3 (Y173) (#ab109544) and total Vav3 (#ab203315) from Abcam, pJNK (Alexa Fluor 647 conjugated, #562481), p-p38 (PE-conjugated, #612565), Rac2 (#610850), pStat3 (#55385) and pStat5 (Alexa Fluor 647 conjugated, #612599) and B220 APC-Cy7 antibody (#552094) from BD Bioscience, anti-phosphotyrosine antibody from Millipore Sigma (#05321), p4EBP1 (PE-conjugated, #12-9107-42) from Thermofisher Scientific.

[0298] Lipids for membrane displacement assays (phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM), and phosphatidylinositol 4,5-bisphosphate (PIP2) were from Avanti Polar Lipids.

[0299] IODVA1 was synthesized from 2-guanidino benzimidazole and purified as described by Gasilina et al. (2020). Imatinib (#SML1027) was from Millipore Sigma, dasatinib (#S1021), and ruxolitinib (#S1378) were from Selleck.

[0300] Production, transduction and transplantation of retroviral and lentiviral particles of transduced leukemia cells: Production of lentiviruses and retroviruses for stable transduction of murine and human cells was performed as previously described (Lee et al., 2017). Scrambled and Vav3-specific shRNAs (Sigma-Aldrich MISSION shRNA) were from the Cincinnati Children's Lenti-shRNA library core. Viral transduction of retroviral and lentiviral vectors, cell lines and murine LDBM and transplantation of transduced leukemia cells have been described previously (Chang et al., 2012).

[0301] For the Vav3 rescue experiment, low-density bone marrow cells from wild-type (Vav3 + / + ) or Vav3 - / - mice were transduced with a bicistronic retroviral vector encoding p190 BCR-ABL1-IRES-YFP (yellow fluorescent protein), and YFP + cells were sorted 48 hours after transduction. Cells were then transduced with lentiviral particles encoding empty vector, full-length Vav3, or ΔCHVav3 (pCDH1-MCS1-EF1-copGFP). Cells were sorted for GFP + / YFP + and treated with the indicated concentration of IODVA1. The cell cycle was analyzed 18 hours after BrdU incorporation.

[0302] SDS-PAGE, pull-down assays, and immunoblotting: Exponentially growing (6x10 6 ) p190-BCR-ABL Ba / F3 cells were treated with vehicle or IODVA1 at the indicated concentrations and time points. Active GTPase pull-downs were completed using GST-PAK1-GBD or GST-Rhotekin (Thermofisher, #16118 and #16116) according to the manufacturer's instructions. Protein complexes were separated on SDS-PAGE and immunoblotted with anti-Rac1, anti-Cdc42, and anti-RhoA antibodies.

[0303] For analysis of expression, cells were lysed in RIPA buffer supplemented with phosphatase and protease inhibitors, separated on SDS-PAGE, transferred to PVDF or nitrocellulose membranes, and blotted with appropriate primary and secondary antibodies as previously described (Lee et al., 2017; Chang et al., 2012). After normalization relative to total protein mass, signals were normalized relative to unstimulated conditions. Quantification was performed using Li-COR Image Studio.

[0304] Cloning: Human Rac1 (GenBank accession number: NM_006908.4) was subcloned into the pFastBacHTB vector (Invitrogen) and fused with an N-terminal His6 tag.

[0305] For pMalX(E)-based expression, full-length Vav3 was amplified with primers overlapping the NotI restriction site of the pMalX(E) vector, which had an N-terminal linker AAAA, AAAASEF, or AAAASEFGS linker. The final construct encoded MBPX(E)-linker-Vav3. For the His6-MBP-N9-TEV-Vav3 construct, full-length Vav3 cDNA with a stop codon at the end of the coding sequence was amplified by PCR using primers overlapping the pET28a-MBP-N9TEV-mOrange vector at the SspI site. The resulting construct encoded MBP-N9-Vav3-His6. All constructs were verified by Sanger sequencing using the CCHCM DNA Core. To minimize aggregation and improve the quality of the purified protein, expression clones were tested with chaperone plasmids according to the manufacturer's protocol.

[0306] Protein expression and purification: For the membrane displacement assay, full-length human Rac1 was purified from baculovirus. pFastBacHTB-Rac1 was transformed into DH10 cells, and the resulting bacmid was used to generate baculovirus in Sf9 cells. Rac1 was produced in TNAO38 insect cells and purified using Ni-IMAC chromatography (Zhang et al., 2014).

[0307] To produce recombinant Vav3, plasmids were co-transformed with the chaperone plasmid Gro7groEL-groES in BL21(DE3) or T7 Express. Cultures were grown in LB supplemented with a metal mix (Studier 2005). Proteins were purified using Ni-IMAC chromatography, dialyzed, and further purified using size exclusion gel filtration (HiLoad Superdex 200 16 / 60). Fractions were analyzed by SDS-PAGE, and protein fractions containing Vav3 were pooled, concentrated to ~10 mg / mL, and flash-frozen in liquid nitrogen. The final yields of MBP-N9-Vav3 and MBPXE-Vav3 were 5 mg and 20 mg per 6 L of culture, respectively.

[0308] Recombinant LARG(DH / PH) was purified as an MBP fusion protein as previously described (Kristelly R. et al., 2003).

[0309] Extraction of prenylated Rac1 from liposomes by RhoGDI: The displacement of prenylated Rac1-GDP from synthetic liposomes by GST-RhoGDI1 in the presence and absence of IODVA1 was investigated using a liposome sedimentation assay as described by Zhang et al., 2014. Briefly, liposomes were generated using a defined lipid composition (194 μg) containing 39% w / w phosphatidylethanolamine, 16% w / w phosphatidylcholine, 36% w / w phosphatidylserine, 4% sphingomyelin, and 5% w / w phosphatidylinositol 4,5-bisphosphate. Prenylated Rac1-GDP (1 μM) was added to liposomes suspended in protein buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 3 mM DTT) and incubated on ice for 20 minutes. In the absence or presence of IODVA1, GST-RHOGDI1 (2 μM) was added to the liposome / prenylated Rac1 and incubated further on ice for 30 minutes. The samples were then centrifuged at 20,000 x g for 20 minutes at 4 °C. The pellet and supernatant fractions were collected, separated on SDS-PAGE, and immunoblotted for Rac1.

[0310] Microscale thermophoresis (MST): Purified Vav3, LARG, or Rac (1 μM) was incubated with the indicated concentration of IODVA1 for 30 minutes at room temperature. Samples were loaded onto a zero-background MST Premium-coated capillary and binding events were measured on a Monolith NT.LabelFree (NanoTemper Technologies). Binding data were analyzed using thermophoresis or thermophoresis with temperature jump analysis as described previously (Jerabek-Willemsen et al., 2011). Binding data were normalized using fraction bound. K d The 95% confidence intervals for the K values were 0.27 to 0.98 μM for Vav3, 5.9 to 10.37 μM for LARG, and 19.6 to 105.8 μM for Rac.

[0311] Stopped-flow spectroscopy. GTPase assays and nucleotide exchange reactions were performed using a Hi-Tech Scientific (SF-61) stopped-flow instrument (Nouri et al., 2016). The excitation wavelengths for tamraGTP and mantGppNHp were 543 nm and 362 nm, respectively. For GTPase assays, equal volumes (600 μl) of 0.2 μM Rac1-tamraGTP and 10 μM p50GAP were used. GTPase assays and protein–protein interactions were performed in the presence of 5% DMSO.

[0312] Animals and in vivo drug administration: Vav3-deficient mice (Fujikawa et al., 2003) and Rac1Δ / Δ+Rac2-deficient (Thomas et al., 2007) mice have been previously described. C57Bl / 6 mice were commercially obtained (The Jackson Laboratory and Harlan Laboratories) and used as donors and / or recipients for transduction / transplantation models according to protocols approved by the Cincinnati Children's Hospital Medical Center Institutional Animal Care and Use Committee. For in vivo drug administration, Alzet osmotic pumps (model 2002, Durect) were used according to the manufacturer's protocol and implanted as previously described (Thomas et al., 2007).

[0313] Histology: Embedded tissues were sectioned into 4-μm slices and then immunohistochemically stained using a mouse-on-mouse kit (MoM kit, Vector Laboratories). Tissue sections were subjected to citrate antigen retrieval, pretreated with 0.3% hydrogen peroxide, blocked according to the kit instructions, and incubated with a phosphorylated Vav3 antibody (dilution 1:200, Abcam) and an HRP-conjugated anti-rabbit secondary antibody (Vector Laboratories). Staining was completed with a DAB peroxidase kit (Vector Laboratories) and counterstained with hematoxylin. Tissue sections were coverslipped with Cytoseal 60 and images were acquired with a Nikon Eclipse Ci microscope.

[0314] CFU-proB assay: B-cell lineage colony-forming units (CFU-proB) were quantified 9 days after leukemia BM cell culture or sorted B-cell progenitors expressing p190-BCR-ABL in M3134 methylcellulose (StemCell Technologies) supplemented with 30% FBS (for murine B lymphoid colony-forming cells; StemCell Technologies), 2 mM L-glutamine (Invitrogen), 1% penicillin-streptomycin (Invitrogen), 100 μM β-mercaptoethanol (Fisher-Scientific), 1% BSA (Sigma-Aldrich), 20 ng / mL recombinant murine IL-7 (PeproTech), and 100 ng / mL recombinant murine SCF (PeproTech).

[0315] Cell cycle analysis: The cell cycle was analyzed by in vitro incorporation of 5-bromo-2'-deoxyuridine using the Brdu Flow kit (BD Pharmingen, catalog number #552598). Briefly, murine leukemia progenitor cells were incubated with 1 mM BrdU solution for 45 minutes, and the cells were further fixed and permeabilized. DNAse treatment was performed according to the instructions and stained with anti-BrdU, and apoptosis was analyzed by 7-AAD staining by flow cytometry.

[0316] Flow cytometry analysis: Red blood cells were removed from peripheral blood samples using lysis buffer without fixative (BD Pharm Lyse lysis buffer, catalog number #555899). After washing once in PBS, the cells were stained with anti-B220 APC-Cy7 antibody. The stained cells were washed once and analyzed by flow cytometry.

[0317] Primary PDX ex vivo drug treatment: Primary patient samples were obtained from patients at CCHMC under protocols approved by the Institutional Review Board (#2008-0021 and #2008-0658). The samples were subjected to RBC lysis, and the isolated WBCs were mixed with OKT3 anti-CD3 antibody to eliminate the possibility of graft-versus-host disease, and then injected into NSG or NRG mice under busulfan conditioning. 10 . The spleen preparations from mice successfully transplanted with B-ALL were co-cultured with MS-5 or OP9 stroma in MEMα medium supplemented with 20% FBS and 10 ng / mL recombinant human SCF (Kit-L), Flt3L, and IL-7 (KF7). IODVA1 was added 24 hours after initial seeding. After 7 days, the co-cultures were collected by trypsin digestion and cell counting was performed using trypan blue. Flow cytometry was performed using mCD45-APC-Cy7 (BD), hCD45-FITC (BD), hCD19-VioBlue (Miltenyi Biotech), and 7-AAd (for viability) to determine the percentage of human ALL in the culture. The total absolute ALL cell number was determined by multiplying the cell count by the percentage of human ALL cells.

[0318] In cell assays, NIRA2 is an effective inhibitor of p190-BCR-ABL-expressing cells. The efficacy of NIRA2 was tested in Ba / F3 cells transduced with p190-BCR-ABL (a commonly used Ph+B-ALL cell model) or the empty vector Mieg3. Ba / F3 cells are bone marrow-derived, interleukin-3-dependent murine pro-B cells that are widely used to study the mechanisms of leukemogenesis and development and the discovery of targeted therapies. The cells were grown in suspension in the presence of IL-3 and NIRA2 (0 to 300 nM), and the cells were counted daily by trypan blue exclusion for 3 days ( Figure 19 ). At 10 and 30 nM, NIRA2 had a cytostatic effect on BCR-ABL-expressing cells. At 100 nM, NIRA2 reduced the viability of BCR-ABL-expressing cells by 98 ± 4% (SEM, N = 9) on day 1; the viability of cells expressing the Mieg3 empty vector was not affected regardless of the NIRA2 concentration. A plot of concentration-cell viability at the 24-hour time point showed that NIRA2 inhibited the survival of Ba / F3 cells expressing p190-BCR-ABL with a half-maximal effective concentration (EC50) of 42.2 nM ( Figure 19 ). Thus, NIRA2 is an effective inhibitor of p190-BCR-ABL-expressing cells but does not affect the proliferation of Ba / F3 cells expressing the empty vector Mieg3, indicating its specificity for cells expressing oncogenes.

[0319] NIRA2 is not a kinase inhibitor. To test whether NIRA2 is a kinase inhibitor, its potential to interfere with the ability of 485 recombinant wild-type and mutant kinases to hydrolyze ATP was evaluated. Each kinase was tested twice at a concentration of 0.5 μM NIRA2 in the presence of ATP (at a concentration of K m or 10 μM) and Mg 2+ (5 mM), and the data were averaged and compared with the vehicle DMSO control. Figure 20 A plot showing the replicates compared to the vehicle control set at 0% is presented. Statistical analysis of the kinase panel data showed that NIRA2 was ineffective against all tested kinases in vitro. It showed 19% to 15% inhibition of MINK1, PKCθ, and MAP3K8 and 30% activation of MYLK2. However, the inhibitory and stimulatory effects were not substantial, and higher NIRA2 concentrations were required to inhibit or stimulate the above kinases to the 50% level. Thus, NIRA2 is not a kinase inhibitor, and given that the cellular EC50 of NIRA2 (42.2 nM) is far lower than the concentrations used in the kinase assays, the cellular effects previously observed in Ba / F3 cells expressing p190-BCR-ABL cannot be explained by kinase inhibition or stimulation.

[0320] NIRA2 inhibits tumor growth in a mouse model of colon cancer. The in vivo efficacy of NIRA2 was tested in a xenograft mouse model generated with murine colon adenocarcinoma MC38 cells. Cells were subcutaneously injected into the lateral side of the hind leg. Each injection contained one million Matrigel + MC38 cells in a volume of 100 μL. Tumors were measured with calipers until they reached an average size of 100 - 200 mm 3 . Animals were divided into treatment groups (N = 10) and administered (5 times per week) via IP 250 μL of PBS solution containing vehicle control, IODVA1 (1 mM), or NIRA2 (0.5 mM). Treatment continued for 2 weeks. Animals were euthanized when the tumor size reached 2 cm 3 or at the end of the study. Tumors were measured daily, and mice were weighed 3 times per week. As Figure 21 shown, both IODVA1 and NIRA2 significantly reduced tumor growth and tumor volume. NIRA2 was more effective than IODVA1, but the dose regimens of both drugs were not optimized. At the end of the study, we did not notice any effect of IODVA1 or NIRA2 on the spleen.

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[0455] Unless otherwise indicated, all percentages and ratios are by weight.

[0456] Unless otherwise indicated, all percentages and ratios are based on the total composition.

[0457] It should be understood that every maximum numerical limitation given in this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were expressly written herein.

[0458] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”

[0459] Unless expressly excluded or otherwise limited, each document cited herein, including any cross-referenced or related patent or application, is hereby incorporated herein by reference in its entirety. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein, or that it alone, or in any combination with any other reference or references, teaches, suggests, or discloses any such invention. Further, in the event that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to the term in this document shall govern.

[0460] Although specific embodiments of the present invention have been illustrated and described, it will be apparent to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the present invention. Accordingly, it is intended to cover all such changes and modifications within the scope of the present invention in the appended claims.

Claims

1. A composition, comprising: and a pharmaceutically acceptable carrier.

2. A composition, comprising: and a pharmaceutically acceptable carrier.

3. A composition, comprising: and a pharmaceutically acceptable carrier.

4. A composition comprising wherein R1, R2 and R4 are independently selected from H, D, halogen, CN, Me, Et or Pr; wherein R3 is mono - substituted or multi - substituted and is H, D, halogen, CN, C1 - C4 alkyl, C1 - C4 alkoxy; wherein each Q is independently selected from N, C; and a pharmaceutically acceptable carrier.

5. The composition according to claim 4, wherein the compound has the following structure: or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

6. The composition according to claim 4, wherein the compound has the following structure: or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of a medicament for treating cancer in an individual in need thereof, wherein the cancer is leukemia.

8. The use according to claim 7, wherein the cancer is selected from chemotherapy - resistant leukemia, immunotherapy - resistant leukemia, recurrent leukemia and other targeted - therapy - resistant leukemia.

9. Use of the composition according to any one of claims 1 to 6 in the preparation of a medicament for treating cancer in an individual in need thereof, wherein the cancer is selected from breast cancer, lung cancer and colorectal cancer.

10. The use according to claim 7 or 9, wherein treating the cancer comprises the following steps: a. determining the level of Vav3 or Rac GTPase in a biopsy tissue obtained from the individual's cancer; and b. administering the medicament to the individual, wherein the level of Vav3 or Rac GTPase is elevated compared to a control.

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