AACR Annual Meeting 2024

Essential biological signaling pathways affecting e.g., proliferation, differentiation, migration, and apoptosis are regulated by protein kinases. Deregulation of protein kinases is observed in many tumor cells and frequently the development and progression of human cancers and other diseases is found to be causally connected to altered activity of specific protein kinases. 

Therefore, protein kinases have become a prime molecular target for therapeutic intervention. Multiple small molecule inhibitors targeting different kinases are currently in clinical use for treatment of various types of cancer and other diseases. 

A first step in preclinical development of new compounds is testing candidate substances in biochemical in-vitro assays, either based on binding of the compounds to their target or based on alterations of the in-vitro activity of the target kinase monitored in biochemical activity assays. 

Depending on the actual mode of action of a compound, the relevance of the results of such in-vitro assays may depend on the actual setup of the assay. 

Mainly for reasons of cost and technical feasibility, the use of generic substrates for in-vitro kinase activity assays is well established. While many of those substrates are highly artificial and can only be considered as generic phosphate-group acceptors, they have been successfully used in the past in the development of many approved small molecule kinase inhibitors currently in clinical use.  

However, even while many kinases may show activity with generic substrates, for some physiological substrates are required. Members of e.g. the RAF family and other members of the MAPK pathway are highly substrate specific and will not show kinase activity with generic substrates. 

Such kinases which have been established with generic substrates are rarely switched to more physiological substrates as most often this will result in increased complexity and associated cost of preclinical compound development. 

We have compared the in-vitro activity of WEE1 using different in-vitro activity assay readouts like autophosphorylation, phosphorylation of generic substrates and phosphorylation of its physiological substrate CDK1. In addition, we examined the substrate specificity of WEE1 towards CDK1 alone and in complex with Cyclin B1. 

We compared the potency of a selection of WEE1 inhibitors when different substrates were used. 

While in early preclinical development the use of rather artificial biochemical assays has been successfully been applied in the past, it should be taken into consideration that at least for the kinase target in focus, the identification, establishment and use of a more physiologically relevant substrate may increase the value of early-stage compound screening results significantly.

Within the last decade, major technological advances in flow cytometry and (single cell) RNA-sequencing have deepened our understanding of complex anti-tumoral immune responses. This allows a comprehensive immune monitoring of novel therapies in pre-clinical models. 

Conventional flow cytometry is reaching its technical limitation. The emitted fluorescence signal of the target population stained with antibodies is evaluated with simple bandpass filters, which leads to spectral overlap and thus the number of parameters that can be analyzed simultaneously is limited. The spectral analyzer technology combines both methods, providing great flexibility in evaluating different immune cell populations. This provides the opportunity to obtain target information by measuring the entire fluorescence spectrum measuring each wavelength individually and thus evaluating the true signal of each fluorochrome unaffected by autofluorescence or spillover. 

Our standard all-in-one flow cytometry panel uses the full capacity of a conventional BD Fortessa flow cytometer and enables the differentiation of important immune cell populations in tumors, such as T cells (CD4+, CD8+, regulatory T cells), B and NK cells as well as macrophages (M1/M2), MDSCs (granulocytes and monocytes) and dendritic cells. However, it would be of great advantage to gain further insights into activation and effector functions of the immune cells using markers like CD44 & CD62L, CD69, PD-1, Lag-3, Tim-3 and PDL-1, CD80, CD86, CD40 and Ki-67. The Sony ID7000 spectral analyzer enables the simultaneous assessment of 27 markers to evaluate immune phenotypes, with the ability to include up to 4 individual target antibodies. 

Flow cytometry data from various tumor models are presented, demonstrating the enormous utility and potential of the spectral analyzer, especially with limited and precious tumor sample material, by evaluating different immune cell populations simultaneously without reaching its technical limits. The spectral analyzer technology enables us to mechanistically investigate differences in tumor therapies such as PD-1 treatment or other immune cell modulators not only phenotypically but also with regard to activation and differentiation of immune cell populations. 

We sought to screen all FDA approved, cancer-indicated, kinase targeting drugs using the gold standard radiometric biochemical activity assay (HotSpot) against the largest collection of kinases in the world (>100 drugs against 735 total kinases including 395 wild type and 340 mutants). Virtually every drug examined exhibits polypharmacology inhibiting many kinases. Rarely does the intended target represent the greatest inhibition observed from a drug. Furthermore, most drugs inhibit a select group of mutant kinases, including especially mutants of kinases distinct from the intended target. Within a given kinase, each cancer-identified mutation is often as distinct as the different wild-type kinases are from each other, in terms of inhibition profile. Considering mutants, then, the cancer-relevant druggable kinome is many thousand distinct targets, not 518 “plus a few”. Exposing this reality is a first step towards developing all the therapeutic tools necessary to ultimately defeat cancer. Here, we reveal vast and specific repurposing opportunities among the existing pharmacopeia for tumors that are driven by kinases not previously recognized as targets for certain drugs, and for tumors that have become chemotherapy-resistant through mutation of specific kinases. The most exciting and clinically relevant opportunities were confirmed in cell-based assays, results of which will also be presented.

The proto-oncogene KRas is a well-described small GTPase that functions as a molecular switch for major physiological signaling pathways involved in cell proliferation, differentiation and survival. It has been shown that activating mutations in KRas are among the most common oncogenic drivers of tumorigenesis. Missense mutations of KRas result in constitutive activation due to impaired hydrolysis of GTP which enhances tumor-promoting downstream signaling pathways. Most KRas mutations are located in exon 2 or 3 including the most frequently altered glycine 12, which is present in most pancreatic cancers as well as in colorectal cancers and lung adenocarcinomas.

A panel of cancer cell lines harboring different KRas mutations such as G12C, G12V or G12D was selected to test KRas inhibitors in several cellular assays: phosphorylation assays, 2D and 3D proliferation assays. Applying the cellular pERK AlphaLISA assay showed high selectivity of several inhibitors for specific KRas mutants, while RAF and MEK inhibitors did not show significant selectivity in the tested cancer cell lines. The KRas inhibitors were then examined in a cellular 2D and a 3D spheroid proliferation assay to determine the inhibitory effect on cell growth. These cellular assays revealed a high selectivity for a specific KRas mutant, which was even more pronounced in the 3D format than in the 2D setup.

The transferability of the cellular data obtained to in vivo was tested in the Hollow Fiber mouse model. The Hollow Fiber model allows the simultaneous study of multiple cell lines implanted in separated drug-(but not cell-) permeable fibers in a single mouse with a study duration of only 16 days. AMG510 (Sotorasib), a drug approved for tumors with a G12C Kras mutation, and MRTX1133, which is currently in clinical phase and shows activity in tumors with a G12D Kras mutation, were screened for their inhibitory effect on the pancreatic tumor cells MiaPaCa-2 (G12C Kras mutation), AsPC-1 (G12D Kras mutation) and BxPC-3 (wt Kras) in the in vivo Hollow Fiber model in female NMRI nude mice. While the growth of BxPC-3 tumor cells was not affected by either inhibitor, the growth of AsPC-1 tumor cells in particular was selectively and significantly inhibited by MRTX1133.

In summary, the in vivo Hollow Fiber model is a fast and cost-effective model that can play an prominent role in drug development as a link between in vitro and in vivo xenograft studies by providing rapid and transferable evidence for in vivo efficacy.

In preclinical research, cell inoculation via bladder wall injection is a standard for studying invasive bladder cancer into the bladder muscle wall. However, a subset of patients presents with non-muscle invasive (superficial) disease. The standard of care for these patients is excision coupled with BCG treatment via catheterization. There is a need to further establish mouse models of cell inoculation via bladder catheterization for early drug development. While the injection places cells into the bladder wall, catheterization coats the inside of the bladder by first instilling Poly-L-Lysine. After instilling Poly-L-Lysine, instillation of luciferase-tagged cell suspension occurs.

MB49-luc cell lines were generated to determine the optimal cell lines for use in analyzing treatments for superficial bladder carcinoma. MB49-luc cells display varying degrees of luciferase activity and growth in vitro. A subset of cell lines were selected for further analysis in vivo, via bioluminescent imaging, survival analysis, flow cytometry and necropsy. The data provides a preclinical reference for the selection of a cell line for analysis of new drug candidates in C57BL/6 female mice inoculated via bladder catheterization.

The RAF protein kinases are key intermediates in cellular signal transduction, functioning as direct effectors of the RAS GTPases and as the initiating kinases in the ERK cascade. In human cancer, RAF activity is frequently dysregulated due to mutations in the RAF family members (ARAF, BRAF, and CRAF) or to alterations in upstream RAF regulators, including RAS and receptor tyrosine kinases. The first and second generations of RAF inhibitors have yielded dramatic responses in malignant melanomas containing BRAF mutations; however, their overall usefulness has been limited by both intrinsic and acquired drug resistance. In addition, cancers with hyperactive RAS exhibit intrinsic resistance to these drugs. In particular, issues related to the dimerization of the RAF kinases can impact the efficacy of these compounds and are a primary cause of drug resistance. We have established biochemical HotSpot™ kinase assay, NanoBRET™ and NanoBIT™ cell assay platforms for High Through Screening of kinase inhibitors. Here, we demonstrate that the 3rd generation of pan-RAF inhibitors LY3009120, LXH254, and Belvarafenib inhibit ARAF, BRAF, CRAF, BRAF(V600E), and CRAF(R391W) kinase activity in biochemical HotSpot™ assay. Our NanoBRET™ target engagement and NanoBITTM cellular assay data show that LY3009120, LXH254, and Belvarafenib bind to KRAS(G12C) primed BRAF and CRAF, and block BRAF and CRAF dimerization. Furthermore, our results show these inhibitors can block the downstream ERK phosphorylation in cellular HTRF assay and induce caspase-3/7 activation in Caspase-Glo 3/7 and Western blot assays in the triple negative breast cancer MDA-MB-231 cells. Taken together, our results indicate the biochemical HotSpot™ kinase activity assay, and NanoBRET™ target engagement and NanoBIT™ cellular assays can serve as great platforms to facilitate RAF drug discovery against human cancers.

Cell surface proteins are increasingly considered in the development of novel anticancer agents, as demonstrated by recent clinical successes in the field of immuno-oncology and antibody-drug conjugates (ADCs). These conjugates specifically target antigens present on the surface of tumor cells, enabling the delivery of cytotoxic agents to specific tumor sites. During the preclinical development of such compounds, for example, when they are tested on a wide panel of cell lines, it is essential to have a rapid and reliable method for monitoring target expression and distribution.

Complementing our 160 Cell line panel ProLiFiler™, we report here the development of a novel method named oncoFLOW-Profiler™ which includes steps of cell fixation, micronic storage, staining, and measurement by flow cytometry. This method is applied in a 96-well format, enabling screening of all cell lines in parallel and analysis of the expression of different targets in just one day.

In this proof-of-concept study, we investigated the level of ERBB2 expression across the ProLiFiler panel of 160-cell lines in order to validate this methodology. We first compared the impact of fixation methods and sample storage on knowingly positive and negative control cell lines over a period of several months. ERBB2 measurements from the oncoFLOW-Profiler™ will be analyzed and validated by comparison with various OMICs datasets from the Cancer DataMiner platform (4HF Biotec), including RNA expression, mutational status, and protein expression levels. To determine the expression cutoff of ERBB2 amplified models, results will be analyzed according to gene copy number variation. Finally, the flow cytometry data generated will be investigated for use as a predictor of response to clinically approved ADCs targeting ERBB2: Kadcyla and Enhertu.

Here we present a method that we have demonstrated is feasible for investigating cell surface receptor/antigens during preclinical phase of drug development. Our method has enabled us to successfully assess ERBB2 in biological samples and to identify models sensitive to ADCs targeting ERBB2. We will also discuss the results of ongoing experiments analyzing the applicability of this screen to intracellular targets and carbohydrate-based surface antigens, which cannot be analyzed either by transcriptomics or proteomics.

Previously we have reported on the relevance of the joint ProLiFiler and Cancer DataMiner platforms when studying the antitumor efficacy of novel antibody-drug conjugates (ADCs), such as Sacituzumab govitecan. In this study, we use our platforms to investigate and compare the selectivity of two FDA-approved Trastuzumab-based ADCs targeting ERBB2: Kadcyla, the first-in-class ADC employing a microtubule inhibitor bound via a non-cleavable linker, and Enhertu, which combines a topoisomerase inhibitor with a protease-cleavable linker. We performed a cell proliferation and survival assay using the ProLiFiler^TM (Reaction Biology) to characterize the in vitro antitumor effects of Enhertu, Kadcyla, and their individual cytotoxins, deruxtecan & mertansine on 160 human cancer cell lines (CLs). The resulting data were uploaded to 4HF´s Cancer DataMiner platform for data analysis. First, we confirmed high potency and limited selectivity of mertansine, as it displayed an IC₅₀ < 250 nM (mean: 27.3 nM) in 95% of cell lines (CLs). A COMPARE analysis confirmed that mertansine data correlated best with those of other microtubule inhibitors in our drug databases (e.g., MMAE, Spearman rho = 0.79, p=9.6E-27). Conversely, a paired analysis showed that CLs were less sensitive to Kadcyla than to mertansine except for CLs overexpressing ERBB2. The 160-CL panel included a total of nine CLs (six mammary, two gastric, one ovarian) with strong ERBB2 overexpression (Affymetrix data >10), mostly associated with ERBB2 gene amplification; seven of them were the most sensitive CLs to Kadcyla (IC₅₀ < 6 nM). Focusing solely on breast cancer cell lines (breast cancer is the approved indication for Kadcyla), we observed a strong correlation between sensitivity to Kadcyla and ERBB2 expression (Spearman r = -0.78, p = 0.003). Interestingly, in line with published data, the ERBB2-overexpressing cell lines SNU-216 (stomach cancer) and JIM-T1 (breast cancer) were less sensitive to Kadcyla. In non-ERBB2-amplified CLs, Kadcyla sensitivity did not correlate with ERBB2 expression levels. Focusing on ERBB2-negative CLs such as hematological CLs, we unexpectedly observed consistent cell inhibition (mean IC₅₀ =33 nM) with Kadcyla, suggesting that the ADC was cleaved, and the payload released into the medium. Overall, this study has demonstrated the relevance of our platforms to study and compare newly developed ADCs. Cytotoxic activities recorded for conjugates and free payloads, along with molecular data for the test cell lines, provides insight in the mode of action of conjugates and their target cell populations and hence can support the development of ADCs. Investigations are underway with Enhertu and its payload deruxtecan, and results will be compared with those obtained for Kadcyla and mertansine.

Over the recent years, knowledge of RNA modifications and their regulation has expanded extensively, providing novel insights and strategies to explore potential therapeutics for pathogenesis of various diseases, including cancer. Several chemical modifications in RNA have been identified so far, among these N⁶-methyladenosine (m6A) modification is the most abundant and well-studied epitranscriptomic marker found in mRNA and long noncoding RNA. Abnormal m6A expression is proven to be associated with tumorigenesis, cancer stemness and drug resistance of cancers. Methylation in 6-Adenosin of RNA is a dynamic and reversible process tightly regulated by its writer, m6A methyltransferase complex and erasers, FTO and ALKBH5. m6A methyltransferase complex contains METTL3-METTL14-WTAP as core components. METTL3 is the catalytic component, which is activated by heterodimer formation with METTL14. High METTL3 expression is found in several cancers such as breast, lung, liver, gastric, colorectal, AML and a METTL3 catalytic inhibitor is found to delay AML progression in mouse models. Protein that binds RNA m6A modification to execute it signaling are known as m6A readers. Readers are categorized into three main classes, YTH domain proteins, IGF2 mRNA-binding proteins and heterogeneous nuclear ribonucleoproteins. YTH family readers are found to have oncogenic roles in several cancers including AML, breast, lung, CRC and glioblastoma. Recent studies suggest inhibition of m6A binding of individual YTH family proteins is a promising therapeutic strategy however, potent inhibitors are yet to be identified. This poster summarizes current assays we have developed to facilitate cancer therapeutic discovery in m6A related protein targets. We present development and validation of an assay suitable identify in METTL3 catalytic inhibitors using our proprietary hotspot technology. We also show development of HTRF based biochemical assays for all five YTH family protein YTHDF1-3 and YTHDC1-2, to screen for molecules that disrupt protein-m6A interaction. We further study the selectivity of an FDA approved drug Tegaserod, a reported YTHDF1 inhibitor by a structure based virtual screening, between YTH family proteins.

The recent advances in cellular immunotherapies have revolutionized the treatment options for hematological malignancies. Among others, genetically engineered T cells to express chimeric antigen receptors targeting CD19 have shown clinical success in patients with B cell malignancies. However, depending on the study design only one to two thirds of the patients experience a complete response upon CD19 CAR T cells treatment. Antigen escape, immune suppressive microenvironment, CAR T cell dysfunction as well as lack of CAR T cell persistence have been discussed to be reasons for the lack of sustained therapy response. Well established models for the preclinical evaluation of cellular therapies are needed for the evaluation of next generation CAR T cells as well as novel combination therapies.

Here, we describe a xenograft animal model of human acute lymphoblastic leukemia (NALM-6 cells) with a suboptimal treatment response to human CD19 CAR T cells from three different donors. First, the activation status and memory phenotype of the CAR T cells from the different donors was characterized by flow cytometry and the anti-tumoral efficacy of the CD19 CAR T cells was evaluated in an in vitro co-culture killing assay using luciferase expressing NALM-6 cells. Next, CD19 CAR T cells from three donors were injected in NALM-6_luc tumor bearing animals. The tumor growth was monitored by bioluminescence imaging and the phenotype of the transferred CAR T cells was checked by flow cytometry.

The frequency of memory T cells differed between the donors whereas the T cells from all donors exhibited all low expression of activation markers. In vitro, all three donors showed high and comparable anti-tumoral killing efficacy independent of their memory status. In vivo, CD19 CAR T cells from all three donors delayed the tumor growth significantly although none of the animals had a complete remission. In line with the individual memory status of the CAR T cells prior to infusion, there were slight differences in the kinetics of the anti-tumoral response. The flow cytometric analysis revealed that the transferred CAR T cells were almost absent in the peripheral blood and spleen whereas for individual animals tumor cells and highly activated CAR T cells were detectable in the bone marrow of the animals.

In summary, the NALM-6_luc xenograft model is a value tool for evaluating next generation cellular therapies and novel combination strategies to overcome the current limitations of cellular therapies.

Proteolysis-Targeting Chimeras (PROTACs) have emerged as a promising strategy for selectively degrading specific target proteins, thereby offering a novel approach to precision cancer therapy. Among the most notable oncogenes, KRAS with the G12C mutation is responsible for 45%–50% of KRAS mutations in non-small cell lung carcinomas. Additionally, BRD4, a well-studied member of the Bromo- and Extra-Terminal (BET) protein family, is implicated in various hematological and solid tumors. The implementation of cell-based screening for PROTACs targeting either KRAS(G12C) or BRD4 represents a highly valuable platform for advancing cancer drug discovery. We utilized the NanoBRET™ Ternary Complex Assay, NanoBiT™ Assay, and Western Blot assay to evaluate the efficacy of PROTACs. The NanoBRET Ternary Complex Assay revealed that KRAS G12C degrader-1 and LC-2 effectively degrade KRAS(G12C), while dBET6 and MZ-1 demonstrated the ability to degrade BRD4. Notably, the NanoBiT assay showed that LC-2 dose-dependently degrades KRAS(G12C) in HEK293 cells co-transfected with KRAS(G12C)-LgBiT and KRAS(G12C)-SmBiT. Moreover, dBET6 exhibited a dose-dependent degradation of endogenous BRD4 in HepG2 cells, and LC-2 demonstrated the degradation of endogenous KRAS(G12C) in MiaPaCa2 cells. In summary, the development of a robust cell-based assay platform for high-throughput screening of PROTACs marks a substantial advancement in cancer drug discovery. This approach not only enables the identification of promising PROTACs but also holds the potential for more effective and targeted interventions in the ongoing battle against cancer.

CAR T cells are donor/patient-derived T cells genetically engineered to express a chimeric receptor against a specific tumor antigen (TA), which allows an MHC-independent recognition and targeting of tumor cells. While CART cell therapies have been tremendously successful against hematological cancers, with 6 T cell products being approved up to date from the FDA (Sengsayadeth S etal, EJHaem. 2022), the activity on solid tumors is more difficult. Solid tumors remain a unique challenge in part due to trafficking problems of CAR T cells, a hostile TME and heterogenous antigen expression, which limits CART cell capacity to find their target. Several strategies are being developed at the moment with the aim to overcome these hurdles, such as multispecific CART cells, co-therapy with checkpoint inhibitors, or inhibition of DNA methylation enzymes to prevent exhaustion. To offer an in vitro platform to assess the effectiveness of upcoming cellular therapies we developed several pipelines to allow the investigation of cytotoxic potential in 2D and 3D formats.

We show here our results using a CART cell product expressing a coreceptor to enhance its activation potential upon recognition of the target. We next tested the capacity of these cells to kill solid tumor in vitro by coculturing with potential target cells cultured in a monolayer (2D). By measuring the luciferase expression of the target cells, we could track the cytotoxicity exerted by the effector cells. In vitro, the CART cells were able to kill in a dose dependent manner different solid tumor cell lines expressing the cognate antigen for the co-receptor. At Reaction Biology we have developed spheroid-based 3D killing assays in which target cells from solid tumor entities are pre-cultured to form spheroids prior to their coculture with effector cells. Using this technique, we challenged the capacity of these CART cells to recognize their antigen in a 3D format. This 3D assay system can be used as well in combination with co therapies such as PD1 inhibitors, in a sensitive and high throughput manner. Finally, we evaluated the therapeutic capacity of these CART cells in vivo in a subcutaneous THP-1 tumor model. Mice treated with these CART cells showed a marked reduction of tumor volume.

In summary we show here a pipeline to characterize in vitro the efficacy of new T cell products, in particular those designed to overcome some of the hurdles against solid tumors.

Macrophage infiltration of the tumor microenvironment is a key process that can determine the success or failure of anti-tumor therapies. Thus, many immunooncology therapies are being developed to modulate the macrophage differentiation program towards the anti-tumor M1 subset, while hindering the M2 subset associated with tumor progression. Monocyte-derived macrophages comprise a crucial in vitro cellular tool to evaluate the plasticity potential between the different polarization states. Several hurdles like their in vitro adherence, low cell number yield and the donor-to-donor variability pose technical challenges when using in vitro differentiated macrophages for compound screening and titration or functional assays.

At Reaction Biology we have established human macrophage assay pipelines to evaluate the novel macrophage polarization compounds in vitro. By isolating monocytes from frozen PBMC stocks it is possible to have access to the same donor or group of donors PBMC for repetitive analysis. We have developed a differentiation protocol that allows us to use different plate format from 6-well to 48 well plates, and thus increase and modulate the throughput according to the required readout.

The effect of therapeutic compounds can be analyzed by flow cytometry. Our data shows that human primary monocytes grown under M1 polarizing conditions express high levels of CD86 and CD80 in contrast to those cultivated under M2 polarizing conditions, with high expression of CD163, CD206, and CD209. Cell culture supernatant can be used to determine cytokine secretion. The functionality of the different macrophage subtypes can be further investigated using a pH-sensitive phagocytosis tracker, like pHRodo-Zymosan or pHRodo-Ecoli, which shows differential uptake according to differentiation profile. Moreover, treated macrophages can be used in coculture with T cells to investigate their capability to foster or inhibit an immune response.

Our data shows the value of monocyte-derived macrophage assays in the evaluation and efficacy assessment of TME modulators.

We sought to screen all FDA approved, cancer-indicated, kinase targeting drugs using the gold standard radiometric biochemical activity assay (HotSpot) against the largest collection of kinases in the world (>100 drugs against 735 total kinases including 395 wild type and 340 mutants). One of these drugs, mitapivat, is an activator of Pyruvate Kinase. Here, we show mitapivat-driven biochemical inhibition of several, but not all, mutants of c-KIT. We also observed the equal and opposite phenomenon whereby many ATP-competitive kinase inhibitors demonstrate biochemical activation of certain kinases, several of which seem particularly susceptible to the effect. Previous work by other investigators (at Genentech and the Rosen lab, MSKCC) demonstrated that PLX4720 (precursor to Vemurafenib) paradoxically activated wild type BRAF, but not BRAF mutants, by driving dimerization of the kinase in drug concentrations insufficient to inhibit both members of the dimerized pair. Our data suggests this phenomenon may be more widespread than previously appreciated and fully observable in vitro. Such paradoxical activation could be driving both toxicities and inefficacy against cancers expressing some of these activatable kinases. This information could suggest novel opportunities for combination therapy and improve outcomes by contraindicating certain medication from use based on tumor expression profile.

Kinesin superfamily proteins (KIFs) are a large family of molecular motor proteins, that share a highly conserved motor domain. KIFs play an important role in cell division and transport of vesicles and organelles within cells. Mitotic kinesins hydrolyze ATP through its ATPase activity to move along the spindle microtubules and carry out several functions during mitosis to facilitate precise chromosome segregation. Altered expressions of several kinesins are shown in various cancers, fueling the aggressive nature of cancers leading to genomic instability. KIF11/Eg5 overexpression is shown in various cancers including hepatic carcinoma, lung, prostate, colorectal, gastric, and pancreatic cancers. Small molecule inhibitors of KIF11/Eg5 and CENP-E are shown to result in mitotic arrest leading to apoptosis in tumor cell lines. Despite the poor clinical outcome of several Eg5 and CENP-E inhibitors entered in clinical trials, targeting kinesins remains an attractive anti-cancer therapeutic approach. Recently, an inhibitor of KIF18A has entered clinical trial for treatment of solid tumors with high chromosomal instability. Here we show development of a microplate based biochemical screening assays for a panel of kinesin motor domain proteins. We summarize kinetic characterization of kinesin proteins and study the selectivity of various previously reported KIF18A, Eg5, CENP-E inhibitors across a kinesin selectivity panel.