CDK12/13 dual inhibitors are potential therapeutics for acute myeloid leukemia

Acute myeloid leukaemia (AML) comprises a group of genetically and phenotypically diverse diseases with unfavourable prognoses. Despite significant advances in genetic profiling, molecular characterization and drug discovery, the treatment of AML has remained stagnant until recently. Since 2017, an increasing number of targeted agents or drug combinations have been approved by the US Food and Drug Administration (FDA) and added to clinical practice, including the BCL2 inhibitor Venetoclax.1 Despite these advances, the outcome is still poor for refractory and relapsed AML patients, and acquired resistance is observed in most patients following targeted treatment. These challenges underscore the need to identify novel therapies capable of overcoming resistance to chemotherapeutics and currently available targeted agents. Cyclin-dependent kinases (CDKs) are a family of 20 different ATP-dependent serine–threonine protein kinases that play an important role in the regulation of gene transcription and cell-cycle progression in response to various cellular processes. CDK12 and CDK13, in particular, have the largest molecular weights (164 and 165 kDa, respectively) and share the highest sequence homology (50% amino acid identity overall and 92% homology in the kinase domain) compared to other members of the CDK family. Recent studies have revealed that both CDK12 and CDK13 form complexes with cyclin K to regulate gene transcription and RNA splicing.2-4 Additionally, there is mounting evidence that CDK12/13 plays a crucial role in the development and metastasis of several solid tumours, such as breast, prostate, ovarian, colorectal and pancreatic cancer, as well as osteosarcoma.5-10 Furthermore, studies have demonstrated that CDK12 is essential for maintaining genomic stability and regulating the DNA damage response.11, 12 Importantly, these studies have also demonstrated that CDK12 inhibition can have a synthetic lethal effect when combined with PARP, MYC or EWS/FLI inhibition, indicating a promising approach to enhance the efficacy of targeted cancer therapy.2-4 Pharmacological inhibition of CDK12/13 has been evaluated as an anti-cancer therapeutic strategy, either as a single agent or in combination with DNA-damaging chemotherapy and checkpoint inhibitors.7, 13, 14 THZ531 and SR-4835 are among the most potent dual inhibitors of CDK12/CDK13, which have demonstrated robust efficacy and selectivity against breast cancer cells.7, 15-17 Aberrant transcription is a hallmark of AML. We, therefore, explore the therapeutic potential of THZ531 and SR-4835 for the treatment of AML. We first analysed the effect of knockout or knockdown of CDK12 and CDK13 by mining CRISPR and siRNA screening data from the Depmap portal. Knockout or knockdown of CDK12 and CDK13 demonstrated growth disadvantages in the majority of myeloid leukaemia cell lines tested with a wide range of gene scores (Supplementary Figure S1). These data indicate that CDK12 and CDK13 are essential genes in leukaemia cell lines and therefore prompted us to test CDK12/CDK13 inhibitors for efficacy in killing leukaemia cell lines and primary patient cells. We examined the effects of THZ531 and SR-4835 against different leukaemia cell lines representing diverse genetic backgrounds. Both compounds demonstrated significant potency against the tested AML cell lines, with IC50s ranging from 34 to 116 nM. However, we observed less effect on the viability of cytokine-dependent cell lines, Ba/F3 and TF-1 (IC50 of 602 and 1301 nM respectively) (Figure 1A and Supplementary Figure S1). We further evaluate the effect of THZ531 on primary AML cells. The response to THZ531 varied greatly across the AML samples, with the most sensitive cases requiring 100- to 1000-fold less THZ531 for effective killing than the resistant cases (Figure 1B, Supplementary Figure S1, and Supplementary Table S1). Interestingly, THZ531 area under the curve (AUC) values exhibited a strong negative correlation with Venetoclax (Figure 1C), suggesting that CDK12/CDK13 inhibitors may be effective in the cases refractory/resistant to Venetoclax-based treatments. We did not observe any correlation between THZ531 and standard-of-care chemotherapy Cytarabine (Supplementary Figure S1). Previously, we have characterized that Venetoclax is relatively resistant to AML samples with FAB M4/M5 morphology.18 In contrast, THZ531 is more sensitive in FAB M4/M5 samples compared to M0/M1/M2 samples (Figure 1D and Supplementary Table S1). We further analysed the RNA expression of CDK12 and CDK13 across different FAB subtypes. Our analysis indicates that FAB M4/M5 samples exhibit relatively lower CDK12 and CDK13 expression (Supplementary Figure S1). This observation may suggest a specific susceptibility of M4/M5 leukaemia samples to further inhibition of CDK12/CDK13 using dual inhibitors. The presented data highlights the contrasting sensitivities of THZ531 and Venetoclax in leukaemia samples and proposes the potential of THZ531 to overcome drug resistance associated with Venetoclax-based therapy. To further validate that CDK12/CDK13 inhibitors are sensitive to Venetoclax-resistant samples, we generated BCL2A1 or KRAS G12D mutant overexpressing as well as TP53 or BAX knockout Molm13 cells, which we and others have discovered confer resistance to Venetoclax and Venetoclax combinations.5, 6, 19 Excitingly, THZ531 and SR-4835 remain sensitive in BCL2A1 or KRAS mutant overexpressing cells, as well as TP53 or BAX knockout cells (Figure 1E). We next obtained samples from patients who had failed Venetoclax/Azacitidine treatment to test the sensitivity of THZ531 and SR-4835. In agreement with the clinically observed response, we found that these samples exhibited high resistance to Venetoclax and/or Venetoclax combinations ex vivo (Figure 1F). Strikingly, THZ531 and SR-4835 were sensitive in all three samples tested (Figure 1F), further validating that CDK12/CDK13 inhibitors can overcome Venetoclax resistance. Since CDK12/CDK13 has a broader effect on leukaemia samples and CDK13 or CDK12 deficiency is lethal during embryonic development, we tested the cytotoxicity of THZ531 and SR-4835 on healthy cord blood-derived haematopoietic stem and progenitor cells (HSPCs). The AUCs of THZ531 for healthy cord blood HSPCs are similar to M1/M2 samples, which are higher than the AUCs of M4/M5 primary patient samples (Figure 1F). These findings suggest that THZ531 and SR-4835 have the potential to selectively eliminate M4/M5 leukaemia cells while sparing normal tissues at effective doses. In summary, our study has demonstrated the efficacy of CDK12/CDK13 dual inhibitors, THZ531 and SR-4835, against both leukaemia cell lines and primary leukaemia samples, including those that are resistant to Venetoclax-based therapies. These results provide a compelling rationale for exploring the use of CDK12/CDK13 dual inhibitors as a potential therapy following the acquisition of resistance to Venetoclax-based combination treatment or as a first-line treatment option for leukaemia samples with Venetoclax-resistant molecular signatures. However, further in vivo and clinical validation is required. Overall, these findings represent a promising direction for the development of new and effective treatments for AML, particularly in cases where Venetoclax-based therapies have proven ineffective. L.S., H.L., R.C, B.A, H.Y.L, and H.Z. performed the experiments. N.L. collected and organized patient sample information, and participated in data analysis and discussion. C.T., S.V.M., and J.W.T. participated in study design, data analysis, discussion, and oversight; H.Z. analysed and interpreted the data and wrote the manuscript, and all authors contributed to the final manuscript. We thank our patients for the generous use of their tissue samples. This work was supported by the Drug Sensitivity and Resistance Network (U54CA224019), and The Cancer Target Discovery and Development Network (U01CA217862) from the NCI. H.Z is additionally supported by a National Cancer Institute R00 (5K99CA237630) grant and Oregon Medical Research Foundation New Investigator Award. J.W.T. was additionally supported by the V Foundation for Cancer Research, The Gabrielle's Angel Foundation for Cancer Research, the Anna Fuller Fund, the Mark Foundation for Cancer Research and the Silver Family Foundation. J.W.T. has received research support from Agios, Aptose, Array, AstraZeneca, Constellation, Genentech, Gilead, Incyte, Janssen, Petra, Seattle Genetics, Syros, Tolero and Takeda. Same as above: samples were obtained with written, informed consent obtained from all patients according to the Declaration of Helsinki and approved Institutional Review Boards (IRB) protocol (Oregon Health & Science University (OHSU) IRB no. 4422; Stanford IRB no. 18329 and 6453). n/a. n/a. Supplementary Figure S1. Supplementary Table S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.