AML Cells Research Articles

Therapeutic Targeting of FLT3 Gate Keeper Mutation with E2082-0047 in Traditional and a Novel Immunocompetent Murine Adoptive Transfer Model of AML

Signaling mutations such as Fms-like tyrosine kinase 3 (FLT3) internal tandem duplication (FLT3-ITD) occur in approximately 30% of AML patients which provide AML cells with a constitutively active aberrant growth advantage, resulting in inferior overall survival. Multiple phase 3 studies with different FLT3 inhibitors validated FLT3-ITD as an important therapeutic target in AML. However, resistance to FLT3-ITD inhibitors occurs via mutations in the tyrosine kinase domain (FLT3-TKD), including the gatekeeper mutation F691L for which all approved FLT3 inhibitors are ineffective. Other mechanisms of secondary FLT3 inhibitor resistance include induction of IRAK4 activity and acquisition of RAS/PTPN11/MAPK activating mutations. New FLT3 inhibitors targeting both intrinsic (FLT3 acquired mutations) and extrinsic mechanisms of FLT3 resistance (e.g. IRAK4 induction) are urgently needed. We demonstrated previously the selectivity and potency of a novel FLT3 inhibitor, E2082-0047, against FLT3-ITD and TKD mutations in vitro using a Ba/F3 cell line harboring different TKD mutations. Guided by KinomeScan data, we demonstrated simultaneous IRAK4 inhibition and verified superior in vivo efficacy compared to gilteritinib using the FLT3-ITD MOLM-13 cell line derived xenograft (CDX) murine model (Elgamal OA et al., ASH 2022). Recognizing the exceptional safety profile of E2082-0047 in pre-clinical IND enabling toxicology studies, we moved forward to test the potency of E2082-0047 versus gilteritinib in cells harboring the FLT3-ITD-F691L gatekeeper mutation using two in vivo models, MOLM-14-F691L CDX model and a novel immunocompetent murine adoptive transfer model that does not require conditioning or irradiation. This model is generated by the crossing of the CD45.2 specific Npm1 cA cre inducible model (Vassiliou et al., 2011) with the Flt3 ITD (Lee et al., 2007) model which was previously reported to harbor a F692L mutation, analogous to the human F691L mutation (Dovey et al., 2016) thus, generating the Npm1 cA;Flt3 ITD-F692L AML murine model. In the MOLM-14-F691L CDX model study, we found a consistent reduction in human CD45 cells in bone marrow and increased survival benefit in mice receiving E2082-0047 compared to gilteritinib. Further, mice in the gilteritinib group with progressing disease (> 50% human CD45+ cells in bone marrow) were switched to E2082-0047 with consequent > 5-fold reduction in bone marrow tumor burden within 8 days. These data suggest that E2082-0047 will be effective in gilteritinib resistant disease. Strikingly, in the AML adoptive transfer model, E2082-0047 resulted in a compelling long-term survival benefit. In our first study (donor #1), vehicle and gilteritinib cohorts had 29- and 32-days median survival, respectively with evident circulating CD45.2 AML. In the E2082-0047 cohort, mice remained on study with an average of <2% circulating CD45.2 cells therefore, we stopped treatment and euthanized three mice for tissue collection, leaving eight mice for disease relapse monitoring for an additional two weeks where we lost 3/8 mice due to early removal criteria (ERC) and 5/8 were euthanized at end of study day 100, with an average of <10% circulating CD45.2 cells. This initial study showed the capacity for E2082-0047 to induce long-term survival in a model with a clinically relevant TKD gatekeeper mutation. We repeated our study with a more aggressive donor (donor #2) where vehicle and gilteritinib arms had a median survival of 22 days. While mice receiving E2082-0047 remain on study (week 8 of an ongoing study), with an average of <5% peripheral blood CD45.2 level (Figure 1). Collectively, our previous and follow-up studies herein demonstrate the superiority of E2082-0047 over gilteritinib against FLT3-ITD and with TKD mutations including the gatekeeper F691L variant. We demonstrate significantly improved survival after E2082-0047 treatment in an immune deficient in vivo model of FLT3-ITD/TKD-F691L MOLM-14 AML and a durable long-term survival in a large subset of mice using two donors in an immune competent adoptive transfer model of NPM1+FLT3-ITD/TKD-F692L AML. Given the curative potential of E2082-0047 in the immune competent model, ongoing efforts are focused on investigating the host immune response to AML. Given the safety and efficacy studies performed with E2082-0047 to date, a healthy volunteer study to best estimate dose for trials in AML is ongoing.

Fc gamma receptors promote antibody-induced LILRB4 internalization and immune regulation of monocytic AML.

The immune checkpoint leukocyte immunoglobulin-like receptor B4 (LILRB4) is found specifically on the cell surface of acute monocytic leukemia (monocytic AML), an aggressive and common subtype of AML. We have developed a humanized monoclonal IgG1 LILRB4-blocking antibody (h128-3), which improved immune regulation but reduced cell surface expression of LILRB4 in monocytic AML models by 40-60%. Interestingly, most of this effect was neutralized by mutation of the Fc region of the antibody (h128-3/N297A), which prevents interaction with Fc gamma receptors (FcγRs). This suggested that there is FcγR-dependent antigenic modulation underlying h128-3's effects, a mechanism known to alter the function of antibodies targeting B-cell malignancies. We disrupted the Fc-FcγR interaction pharmacologically and with stable CRISPR-Cas9-mediated genetic knockout of FcγRs in monocytic AML cell lines to investigate the role of FcγR-dependent antigenic modulation in the regulation of LILRB4 by h128-3. When FcγRI is inhibited or removed from the surface of monocytic AML cells, h128-3 cannot optimally perform its blocking function, resulting in activation of the LILRB4 inhibitory receptor and leading to a 15-25% decrease in T-cell-mediated cytotoxicity in vitro. In the absence of FcγRI, scaffolding by FcγRIIa allows h128-3 to maintain LILRB4-blocking function. Here we define a FcγR-dependent antigenic modulation mechanism underlying the function of an immunoreceptor blocking antibody for the first time in myeloid malignancy. This research will facilitate the development of safe, precision-targeted antibody therapeutics in myeloid malignancies with greater potency and efficacy.

Phase 1 Trial of Dendritic Cell/AML Fusion Cell Vaccine after Allogeneic Stem Cell Transplant in Acute Myeloid Leukemia

Introduction We have developed a vaccine in which patient-derived AML cells are fused with donor-derived dendritic cells (DCs). Vaccination after initial remission and consolidation was shown to elicit leukemia-specific immune responses. We hypothesized that donor-derived DC/AML vaccination post-transplant would elicit the durable expansion of leukemia-specific T cells within the donor T cell repertoire to effectively protect against disease relapse. We report updated results of a phase 1 clinical trial (NCT03679650) evaluating the use of DC/AML fusion vaccine following allogeneic transplant. Methods DC/AML fusion vaccine was generated with patient leukemia blasts, cryopreserved at the time of diagnosis, and donor-derived DCs, from leukapheresis of patients who achieved hematopoietic recovery and remained in CR 25-90 days after an allogeneic transplant. DCs were differentiated via culture of adherent mononuclear cells in the presence of GM-CSF, IL-4 and TNFa. Vaccine was administered starting day 70-100 post-transplant as long as the patient did not have active GVHD requiring therapy. Two doses of the vaccine were given subcutaneously at 3 week intervals, in conjunction with 100 mcg GMCSF daily at the vaccine site for 4 days. A booster vaccine was given 30-60 days following the taper of immune suppression, in the absence of GVHD. Longitudinal profiling of the immune landscape by single cell RNA sequencing and CyTOF from peripheral blood samples obtained before and after vaccination is underway. Immunologic response of leukemia-specfic T cells is being evaluated via IFN-gamma expression following stimulation with autologous tumor lysate. OS was calculated using Kaplan Meier Curve. Results 27 participants have been enrolled with median age 59 years (range 23-74). 15 patients were transplanted with a matched unrelated donor, 10 with a matched sibling donor and two with a haplo-identical donor. One patient relapsed prior to leukapheresis and was ineligible. The median yield of leukemia cells was 218x10 6 (range 62-818x10 6)and mean viability was 97%. The median yield of DCs was 106x10 6 (range 19-182x10 6) and mean viability 81%. Fusion vaccine was successfully generated in 25 of 26 patients who underwent leukapheresis; one patient had insufficient DC. Mean fusion efficiency was 47% with viability of 79%. Mean fusion vaccine dose was 3.8 x10 6 cells. 6 patients from whom vaccine was generated did not meet eligibility to initiate vaccination due to GVHD or COVID19 infection. 19 participants initiated vaccine administration and are evaluable for toxicity and immunologic response. The most common side effects were vaccine site reactions (n=10 grade 1, n=2 grade 2). 5 patients developed GVHD that was attributed as possibly related to vaccination, at a median time of 17 days after vaccination (range 5-70 days). Acute GVHD included two events of skin grade 2, one GI grade 2, one liver and skin grade 3. Chronic GVHD included a moderate event affecting 3 organs and severe event affecting 4 organs. There were 10 additional GVHD events with a median time of 108 days post vaccination (range 7-126 days), assessed as unlikely related or unrelated to vaccine based on pre-existing GVHD and timing of onset. Median time of follow-up is 23 months post-transplant (range 11-59 months). Amongst 19 vaccinated patients, 14 remained in CR and median overall survival estimate is 49 months post transplant. 3 died of relapsed disease, 2 died of GVHD and/or infection, and 1 died of unknown cause. Preliminary results of PB CyTOF from 4 patients in remission and 1 patient that relapsed indicate higher proportions of T cells and DCs in patients in CR. Interferon expression was higher in a patient with durable remission while absent from a patient with early relapse. Conclusions Vaccination using host-derived leukemia cells and donor derived DC was safe and feasible. Adverse events post vaccination have included injection site reactions as well as acute and chronic graft versus host disease. Vaccination may be a promising strategy to reduce relapse as 14 of 19 vaccinated patients remain relapse free. Albeit this approach is limited to patients who have not had early relapse or early GVHD. Immunologic response by interferon release upon lysate stimulation, CyTOF and single cell RNA sequencing of the immune milieu will be reported.

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Inhibiting Drp1 Dependent Mitochondrial Fission

Background: Primary or secondary resistance to venetoclax is frequently associated with mutational/non-mutational activation of MAPK pathways. ERK1/2 are terminal kinases in MAPK pathway and may be an appropriate target regardless of the upstream mechanisms that activate the pathway. ERK1/2 mediated phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth in RAS driven solid cancers (Kashatus et al., Mol Cell. 2015). However, the role of Drp1 dependent mitochondrial dynamics in therapeutic resistance in AML is unexplored. Methods: ERK1/2 was inhibited using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018), an analog of ASTX029 (Munck et al., Mol Cancer Ther. 2021) in vitro and using ASTX029 in vivo. Preclinical models of venetoclax resistance and primary patient samples (n=8) were used to assess the synergy of concomitant Bcl2 and ERK1/2 inhibition (ERKi). In addition, a comprehensive analysis of alteration of signaling pathways, apoptotic signatures and DNA damage responses in response to ERKi+/-venetoclax were analyzed by mass cytometry based proteomic analysis (CyTOF) and immunoblotting. The potential clinical relevance of ERK1/2 inhibition to overcome venetoclax resistance was confirmed in a PDX model of AML (established from an AML patient who relapsed after venetoclax/decitabine treatment). Mitochondrial images were acquired using super-resolution imaging with OMX-Blaze followed by quantification with Imaris. Results : We previously reported the synergy of ERK1/2 inhibition using Compound 27 with venetoclax at inducing apoptosis in RAS mutated and/or venetoclax resistant AML cells including venetoclax resistant isogenic lines (Sharma et al., Blood 140; Supplement 1, 2022). Venetoclax+ERKi depleted leukemia progenitor cells in primary AML samples (CI:0.03-0.23) and impaired clonogenic growth of NRAS mutant PDX cells. In a PDX mouse model of post venetoclax/decitabine-relapsed AML, ASTX029+venetoclax treatment improved survival compared to vehicle (median survival 76.5 days vs. 50 days, p=0.0006) and venetoclax alone (median survival 76.5 days vs. 51.5 days, p=0.0065) (Figure 1) with corresponding reduction in leukemia burden in bone marrow (p<0.0001) and spleen (p<0.0001). CyTOF analysis using PDX bone marrow showed decreased expression of Mcl-1 and pMcl-1-T163 and an increased expression of BIM in response to ERKi+/-venetoclax (Figure 1). To maintain stemness in AML, mitochondrial ROS mitigation and Drp1-mediated mitochondrial fission are crucial (Schimmer et al., Cell Stem Cell. 2018). The inhibition of ERK1/2 resulted in decreased pDrp1-Ser616, along with an increase in mitochondrial length (p<0.001) suggesting impaired mitochondrial fission. This was accompanied by a decrease in mitochondrial membrane potential (p<0.0001) and an increase in mitochondrial ROS (p<0.0001). Overexpression (OE) of a phospho-mimetic i.e. Drp1-Ser616Glu-Ser637Ala led to shorter mitochondrial length (Figure 2), suggesting enhanced fission, a distinct metabolic phenotype with decreased ROS production and decreased mitochondrial depolarization with venetoclax+/- ERKi. Finally, Drp1 phospho-mimetic OE reversed apoptosis induction by venetoclax +/- ERKi (Figure 2) as compared to the wild-type and phospho-null (Ser616Ala) Drp1 (p<0.001) expressing cells, supporting the role of mitochondrial fission in resistance to venetoclax. Conclusion: The increased mitochondrial fission driven by ERK1/2 mediated phosphorylation of Drp1 contributes to venetoclax resistance in AML and inhibiting ERK1/2/Drp1 axis overcomes resistance to venetoclax by inhibiting mitochondrial fission (Figure 2). These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in the treatment of AML.

Loss of Function MBD4 Mutation Impairs DNA Repair in Association with TET2 in Acute Myeloid Leukemia

Background: Methyl-CpG binding domain 4 (MBD4) is a known DNA glycosylase involved in critical steps of base excision repair (BER) and DNA mismatch repair (MMR) and is essential for reducing genotoxic stress in normal cells. MBD4 binds to 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) - oxidation products of TET2 dioxygenases - and may initiate a multi-step BER/MMR around mCpG/hmCpG DNA. Loss of function (LOF) mutations in MBD4 (MBD4 MT) result in compromised BER/MMR, leading to genomic instability. Certain germline MBD4 variants have been reported to be predisposing factors for early onset AML, among other cancers. 1,2 In cases screened at our institution, biallelic MBD4 p.R431* & p.L563* tracked with AML in brother and sister. 3 This inspired us to hypothesize that partial or complete loss of MBD4 may result in reduced BER/MMR function and ultimately in genomic instability and faster accumulation of somatic lesions favoring malignant evolution. Methods: We performed NGS and epigenetic studies coupled with detailed biochemical analyses in CRISPR engineered cell lines and primary AML cells and in vivo model systems to dissect the mechanism of clonal evolution of MBD4 MT hematopoietic stem and progenitor cells (HSPCs). Results: We analyzed WES data from 1,692 patients with myeloid neoplasms (MN, 732 MDS; 960 AML). After adequate filtering based on frequency in healthy population (odds ratio of >1.5), 16 variants in MBD4 were identified in 24 MN cases (MDS=9, AML=15). Among them, 4 heterozygous variants of unknown significance of suspected germline nature (p.R425Q, p.N461S, p.R486K, p.D504H) mapped in the glycosylase domain. Comparing MBD4 MT vs. wild-type ( MBD4 WT) cases, MBD4 MT were found in younger patients (median 55 vs. 71 yrs; p = 0.012) with TET2 MT co-occurring at a lower frequency (8 vs. 24%). Since MBD4 is known to preferentially bind with 5hmCpG DNA, a stable epigenetic mark induced by TET2, we investigated the biochemical basis of MBD4 dysfunction and its connection to LOF TET2 MT. We show that MBD4 hydrolase activity levels affect the genomic accumulation of 5hmC in a TET2-dependent manner. MBD4 KO led to more than 2-fold increased 5hmC in TET2WT cells but not in TET2KO cells, suggesting a functional connection between MBD4 and TET2 in BER/MMR. To test the function and effects of the 4 variants (p.R425Q, p.N461S, p.R486K, p.D504H) identified in our cohort on the activity and function of MBD4, we generated these mutations in a pcDNA plasmid using site directed mutagenesis. The mutants were ectopically expressed in isogenic CRISPR-engineered MBD4 KO HEK293 cells and its effects on 5hmC accumulation were tested. The impact on the functional outcomes were compared to WT controls as described earlier. 4 We found that these 4 variants lead to MBD4 protein instability, and thereby lower MBD4 DNA glycosylase activity resulting in increased (1.6 to 3.3-fold) genomic accumulation of TET2-dependent oxidation products. To establish the effect of MBD4 MT in primary cells, we generated CRISPR-engineered human CD34 + HSPCs and assessed their clonal evolution via colony forming assay. Loss of MBD4 led to clonal advantage reflected in persistent colonies in MBD4KO CD34 + HSPCs that demonstrated myeloid differentiation bias similar to what was observed in patients, suggesting that MBD4 MT may favor clonal evolution to fully blown MN. Finally, transcriptomics analysis of MBD4KO in K562 cells in the presence and absence of functional TET2 demonstrated loss of BER genes compared to WT. The co-immunoprecipitation of MBD4 followed by western blot analysis identified XRCC1, a key component of the BER complex, as a part of MBD4 interactome. Comprehensive characterization of protein complexes with LCMS/MS in K562 cells suggests that MBD4 is involved in maintaining genomic integrity. Consistent with our hypothesis, MBD4 KO in K562 and THP1 cells resulted in a 2- and 3-fold increase in spontaneous mutations respectively, as observed via HPRT assay. Conclusions: Genomic instability due to MMR/BER dysfunction in MBD4 MT cells may result in a higher rate of clonal progression. Interestingly, MBD4 deficiency may provide opportunity for therapeutic targeting of DNA repair pathways in a subset of MBD4 MT-associated MN. Indeed, our findings indicate the possibility of leveraging MBD4 as an actionable target to regulate DNA methylation in association with TET2 during leukemogenesis.

Selectivity and Safety of VIP943: A Novel CD123-Targeting Antibody-Drug Conjugate (ADC) Using a Proprietary Linker and Payload Class

Introduction ADCs afford high efficacy though often have shortcomings in their safety profile. Most ADC payloads are cytotoxic agents that can inhibit general processes of cellular homeostasis, including microtubule polymerization or DNA damage repair. While these are good targets for cytotoxicity, they have the drawback of being nonselective for dividing cancer cells. Here, we report the potent, on-target activity and selectivity of our novel CD123-targeting ADC VIP943, which uses a kinesin spindle protein (KSP; also known as KIF11 or Eg5) inhibitor as the payload (VIP716). The kinesin superfamily (KIF) is comprised of motor-proteins involved in various cellular mechanisms, including formation of the bipolar mitotic spindle during cell division. Targeting KSP selectively inhibits dividing cells, while sparing nondividing, differentiated cells thereby increasing the therapeutic index of VIP943. Methods VIP716 selectivity and potency: The motor domain of the human KSP and the other KIF family members were incubated with stabilized microtubules in a biochemical assay. The freshly prepared mixture was then incubated VIP716 and ispinesib, a clinical stage KSPi. ATPase activity was directly analyzed by measuring the formed inorganic phosphate. VIP943 selectivity: The binding of VIP943 to 6,019 human plasma membrane proteins expressed on HEK cells was evaluated (Retrogenix cell microarray). Cell cycle/death analysis: The effect of VIP943 on the 3 phases of the cell cycle (G0/G1- S- G2/M) was measured with DNA staining and flow cytometry in MOLM-13 and MV-4-11 cell lines. In addition, cell death was measured by flow cytometry after a 4 h incubation with VIP943 in MOLM-13 cells. Isotype ADC was used as a control. In vitro cytotoxicity: 56 hematologic cell lines were treated in vitro with VIP331 (cell permeable analog of VIP716). IC 50 results were matched with the DNA/RNA profile of relevant oncogenes to determine correlations. Nonhuman primate safety and toxicokinetics: VIP943 was intravenously administered to Cynomolgus monkeys at 1, 3, or 9 mg/kg once weekly for 4 weeks in a Good Laboratory Practice (GLP) toxicology study. Serial plasma samples were collected on Day 1 and Day 22. Total antibody from VIP943 (as a surrogate for VIP943) and VIP716 concentrations were measured by LC/MS/MS. Toxicokinetics (TK) were assessed using noncompartmental methods. Results In a biochemical assay, VIP716 (payload), achieved comparable potency to ispinesib with IC 50 values of 1.4 and 1.6 nM, respectively, for KSP. VIP716 did not inhibit any of the 11 other KIF family members up to 10 µM. In the Retrogenix assay, VIP943 (ADC) had no interaction with any of the analyzed human cell surface proteins except for CD123. Cell cycle analysis in AML cells showed a dose- and time-dependent increase in G2/M arrest with VIP943 treatment, but not with isotype control. An increase in cell death with VIP943 treatment was also observed after a 4-hour incubation. In a broad hematologic cell line panel, the median cytotoxicity IC 50 for VIP331 (cell permeable KSPi) was 2.49 nM. Of note, no genomic alterations correlated with sensitivity, suggesting VIP943 may be effective even in hard-to-treat hematologic malignancies. VIP943 was well tolerated in the GLP monkey toxicology study. No mortality occurred in any monkeys. No neutropenia, thrombocytopenia or anemia were observed. VIP943 and VIP716 exposures increased with increasing dose in a dose-proportional or greater than dose-proportional manner. No consistent differences in TK were observed with sex. Based on area under the concentration curve (AUC), no accumulation was observed. Payload to parent ratios were consistent with a low level of non-specific release of VIP716 from VIP943. Conclusion High on-target activity of VIP716, the payload of our new CD123-targeting ADC, VIP943, was demonstrated in a biochemical assay and selectivity against other KIF family members was confirmed. This activity translated into cell cycle arrest in the G2/M phase and subsequent cell death in a dose- and time-dependent manner. Additionally, toxicology in non-human primates and in vivo TK studies confirm safety, favorable drug exposures, and little non-specific release of the payload. Based on these data, evaluation of VIP943 in human clinical trials is warranted.

Combining Allogeneic Gamma Delta T Cells with a Phosphoantigen Prodrug Promotes Apoptosis of Acute Myeloid Leukemia Stem and Progenitor Cells

Despite recent therapeutic advances, patients with relapsed/refractory acute myeloid leukemia (r/r AML) have limited treatment options and poor prognosis. Leukemic stem cells (LSCs) are considered as relapse-initiating cells and the “culprit” of treatment resistance. Allogeneic and autologous gamma delta (γδ) T cell-based therapies have been explored for a long time and proven to be safe in patients through multiple clinical trials. However, quite a few AML blasts obtained from r/r AML patients show resistance to γδ T cell-mediated cytotoxicity. Novel strategies targeted LSCs may improve efficacy of γδ T cell-mediated r/r AML therapy. Given that γδ T cells exert antitumor capacity via multiple pathways, to identify the way matters most in the resistance of γδ T cell-mediated cytotoxicity, we examined the median fluorescence intensity (MFI) of the ligands expressed in tumor cells by flow cytometry, including butyrophilin 3A (BTN3A), butyrophilin 2A1 (BTN2A1), the human MHC class I chain-related genes (MICA and MICB), the unique-long 16 binding proteins (ULBP1, ULBP2/5/6, ULBP3), Fas, the receptor of TNF-related apoptosis-inducing ligand (TRAIL) DR5, CD112 and CD155. The cytotoxicity activity of γδ T cells was measured by flow cytometry-based killing assay or bright-lite luciferase assay system after coculturing with AML cell lines and blasts at an appropriate effector to target (E:T) ratio. Then we employed a Support Vector Machine model to predict the killing efficiency and identified that BTN3A and BTN2A1 play the predominant role in γδ T cell-mediated cytotoxicity based on the Shapley additive explanation. BTN3A and BTN2A1 are the key ligands in the γδ TCR-phosphoantigen (pAg) recognition activity. PAgs, which are produced in tumor cells function as “molecular glues” to promote heteromeric association of BTN3A1 and BTN2A1 (Yuan L et al., BioRxiv 2022). To promote the γδ T cell-mediated cytotoxicity targeting r/r AML, here we synthesized a pAg prodrug, which show great plasma stability and membrane permeability. We anticipate this small molecule would lead pAg accumulation in tumor cells and cause increased γδ T cell activation. To identify the killing efficacy of pAg prodrug-induced γδ T cell activation, we select KG-1α (a human leukemia stem cell-like cell) as target cell. Zoledronate (ZOL), which is employed to stimulate γδ T cells in multiple γδ T cell-based clinical trials, is used in our killing assay as compared group. As expected, combining γδ T cells with pAg prodrug or ZOL both significantly improved the percent of specific killing when the E:T ratio is 1:1. The pAg prodrug showed more potent activity than ZOL group with EC50 value of 0.1 nM vs 17.93μM. And the specific killing percent of pAg prodrug group is 83.26±2.1 % (1 nM), comparing with negative control 15.17±3.4 % (only γδT cells). Futhermore, at relatively low E:T ratio (1:4), γδ T cells with pAg prodrug remain excellent cytotoxicity when targeting MV411 cell and OCI-AML3 cell, which are widely used in r/r AML research. After coculturing with primary leukemia stem and progenitor cells (CD34 +) obtained from r/r AML patients for 6~8 h, the apoptosis percentage of CD34 + leukemia cells was 42.34% comparing with negative control (only γδT cells) 2.53%. Then we evaluated the in vivo efficacy of combining γδT cells and pAg prodrug in NSG mice bearing MV411 cells. Compared with only γδT cells-treated group, combination with pAg prodrug presented better tumor clearing. To elucidate the pathway of pAg prodrug-mediated killing, we tested mitochondria apoptosis related events. After treated by γδ T cells with pAg prodrug, KG-1α cells showed loss of mitochondrial outer membrane potential and increasingly actived caspase 3/7. Meanwhile, pAg prodrug directly activates effector γδ T cells with increased secretion of CD107a and IFN-γ. Considering the possibility of toxicity towards normal hematopoietic stem and progenitor cells, we conducted colony-forming units (CFUs) assay. After treatment with γδT cells and pAg prodrug, the number of CFUs of CD34 + cells from cord blood was not effected compared with the CD34 + cells only group. Overall, we explored the predominant factor in γδ T cell-mediated killing and synthesized a pAg prodrug to improve the killing efficacy. We demonstrated that combining γδ T cells with pAg prodrug has effectively promoted apoptosis of AML stem and progenitor cells sparing normal hematopoietic stem and progenitor cells.

Preclinical and Clinical Evidence for Erythroid-Stimulating Activity of RVU120 CDK8/19 Inhibitor in AML and MDS

Introduction: RVU120 is an orally bioavailable small molecule inhibitor targeting mediator complex kinases CDK8 and its paralog CDK19. Preclinical results indicated that treatment with RVU120 could reduce the viability of AML cells, in particular those positive for NPM1 and DNMT3A mutations. Further studies also indicated that RVU120 induces erythroid differentiation of malignant stem cells isolated from AML and MDS patients, likely by the induction of STAT5-GATA1-dependent transcription. The first signs of clinical efficacy have been observed in the ongoing Phase 1 dose escalation study of RVU120 in AML and HR-MDS patients. The presented work provides the preclinical rationale and growing clinical evidence that in addition to previously reported blast reduction, RVU120 has also strong erythropoiesis stimulating activity that may be useful as a potential therapeutic strategy for MDS. Methods: RVU120-induced erythroid differentiation was studied in primary malignant stem cells from MDS patients and in a stem cell model derived from cord blood cells transformed by TLS-ERG fusion. Erythroid differentiation was tracked by flow cytometry, chromatin status, and transcriptomic changes using RNAseq, ATACseq and ChIPseq. CLI120-001 study (NCT04021368) is a phase 1 dose escalation study of RVU120 in patients with relapsed/refractory AML and HR-MDS which is currently ongoing. Flow cytometry for erythroid differentiation markers, NGS myeloid panel and bulk RNAseq were performed in all patients enrolled after September 2021. Results: At the time of the abstract submission, within 24 evaluable patients' erythroid improvement was reported in 1 patient with HR-MDS, 3 patients with AML MRC, and 1 patient with AML secondary to MF. Interestingly, in these cases, erythroid improvement was observed independently of significant blast reduction, at doses from 25 mg to 135 mg every other day in 3 weeks cycles. Transient induction of erythroid differentiation markers CD71 and/or CD235a was confirmed in 3 additional transfusion-dependent patients treated with RVU120. Bulk RNA-seq confirmed broad transcriptomic changes in BM of selected patients after treatment compared to the pre-dose baseline levels. Robust induction of genes involved in erythroblast differentiation and hemoglobin metabolism genes was confirmed in 2 AML-MRC and 2 AML patients. These changes correlated with the repression of major pro-inflammatory pathways, including hallmark interferon gamma and TNF/NFKB response genes and repression of TGFβ pathway genes. Molecular and flow cytometry changes observed in patients were consistent with the erythroid commitment and sequential induction of erythroid differentiation markers in cultured CD34+ malignant stem cells isolated from MDS and AML-MRC patients treated with RVU120 and other chemically non-related CDK8 inhibitors. Conclusions: The presented results provide clinical and preclinical evidence for RVU120 as a candidate for a novel erythroid stimulating agent. Treatment with RVU120 could be a promising addition to the current treatment options for patients with lower-risk MDS who are transfusion-dependent and failing first-line therapies.

Safety and Efficacy of Selinexor Sequential Azacytidine in Newly Diagnosed Patients with Myelodysplastic Syndromes EB1 or EB2: A Single-Center, Single-Arm, Phase Ib/II Trial

Background: Azacytidine is one of the hypomethylating agents (HMAs) for the treatment of higher-risk myelodysplastic syndromes (MDS). However, clinical outcomes of patients treated with azacytidine monotherapy were far from satisfactory with overall response rate (ORR) of 41%-59% and overall survival (OS) of 15-18 months. Selinexor is an oral, first-in-class selective inhibitor of nuclear export compound that inhibits exportin 1 (XPO1). Preclinical studies have shown that inhibition of XPO1 causes nuclear accumulation of p53 and disruption of NF-κB signaling, both relevant targets for myelodysplastic syndromes. Prior study reported an ORR of 26% and mOS of 8.7 months with selinexor in HMAs-refractory MDS patients. We therefore aimed to assess the safety and efficacy of selinexor sequential azacytidine in newly diagnosed patients with MDS-EB1 or EB2. Methods: The effectiveness of simultaneous and sequential treatment of azacytidine and selinexor was measured by cell death rate, cell cycle and colony formation rate in vitro. Western Blot was performed to detect the sublocation of p53. MOLM-13-luciferase xenograft murine model was established to verify the synergetic effect in vivo. Based on the experiments, we conducted a single-center, single-arm, phase Ib/II trial, which included newly diagnosed patients 18 years or older with MDS-EB1 or EB2. Eligible patients received 2-4 weeks long cycles of oral selinexor (40mg qw for 4 weeks; 40mg biw for 2 weeks; 60mg qw for 4 weeks). The primary endpoints were to find the recommended phase 2 dose (RP2D) and maximum tolerated dose (MTD). The secondary endpoint was ORR including complete remission (CR), partial remission (PR), marrow complete remission (mCR), and hematological improvement (HI). This trial was registered with the Chinese Clinical Trial Registry (#ChiCTR2200065167). Results: First we treated three AML cell lines: AML-OCI2, THP1, MV4-11 and three MDS cell lines: SKM-1, MOLM-13, MDSL with azacitidine and selinexor simultaneously or sequentially. Sequential treatment showed stronger synergetic effect (Figure 1A) and reduced colony formation by 90% in SKM-1 and MOLM-13 (Figure 1B). Cell cycle arrest were induced and the percentage of Annexin V cells obviously increased with sequential treatment (Figure 1C). Sequential treatment decreased leukemia burden and prolonged survival of MOLM-13-luciferase xenograft murine model (Figure 1D, E). Mechanistically, pretreatment of azacitidine on SKM-1 and MOLM-13 upregulated the expression level of p53, and selinexor led to massive nuclear accumulation of p53, resulted in better vulnerability lethal to MDS cells (Figure 1F). Secondly, 15 patients were enrolled in our trial with a median age of 57. 26.7% of patients were MDS-EB1, 73.3% were EB2. 93.3% of patients were high or very high risk by the Revised International Prognositic Scoring System (IPSS-R) (see the Table). At Phase Ib, the following selinexor dosages were administered: 40mg qw (n=3), 40mg biw (n=3) and 60mg qw (n=3). One patient in the 60mg qw group discontinued treatment because of nausea, vomiting, fatigue and Herpes simplex virus infection. Subsequently, in order to find RP2D, an extension cohort of 3 additional patients were treated with 40mg qw and 40mg biw, respectively. Therefore, 14 patients were evaluable for efficacy assessment. ORR was 78.6% in 11 patients including CR in 3 patients (21.4%); mCR+HI in 1 patient (7.1%); and mCR in 7 patients (50.0%) (see the swimmer plot). Especially, among the TP53 mutated patients (n=3), one achieved CR, the other two achieved mCR. The common adverse events were neutropenia (93.3%), anemia (86.7%), thrombocytopenia (80.0%), fatigue (53.3%), nausea (26.7%), dizziness (26.7%), vomiting (20.0%) and decreased appetite (20.0%). There were no treatment related deaths. Conclusion: We verified the activity of selinexor sequential azacytidine in MDS both in vitro and in vivo. The treatment showed with encouraging efficacy in newly diagnosed patients with MDS-EB1 or EB2. Adverse events were manageable with supportive care implementation. Enrollment is ongoing to assess the efficacy and safety of this sequential therapy, which may be a new treatment option for EB subjects.

A Phase I Trial of Sirolimus with "7&3" Induction Chemotherapy in Patients with Newly Diagnosed Acute Myeloid Leukemia.

Chemotherapy remains a primary treatment for younger AML patients, though many relapse. Data from our group have shown that highly phosphorylated S6 in blasts may predict response to sirolimus given with chemotherapy. We report the results of a phase I study of this combination in newly diagnosed AML and the pharmacodynamic analysis of pS6 before and after treatment. Subjects received sirolimus (12 mg on day 1, 4 mg daily, days 2-10), then idarubicin and cytarabine (days 4-10). Response was assessed at hematologic recovery or by day 42 using a modified IWG criteria. Fifty-five patients received sirolimus. Toxicity was similar to published 7 + 3 data, and 53% had high-, 27% intermediate-, and 20% favorable-risk disease. Forty-four percent of the high-risk patients entered into CR/CRp. Seventy-nine percent of the intermediate-risk subjects had a CR/CRp. All favorable-risk patients had a CR by day 42; 9/11 remained alive and in remission with a median follow-up of 660 days. Additionally, 41/55 patients had adequate samples for pharmacodynamic analysis. All patients demonstrated activation of S6 prior to therapy, in contrast to 67% seen in previous studies of relapsed AML. mTORC1 inhibition was observed in 66% of patients without enrichment among patients who achieved remission. We conclude that sirolimus and 7 + 3 is a well-tolerated and safe regimen. mTORC1 appears to be activated in almost all patients at diagnosis of AML. Inhibition of mTORC1 did not differ based on response, suggesting that AML cells may have redundant signaling pathways that regulate chemosensitivity in the presence of mTORC1 inhibition.

Generation and characterization of iPS cell line (SYSUSHi002-A) from PBMCs of an AML patient with TP53 mutation

We have generated an iPSCs line (IPS-AML2-C3, SYSUSHi002-A) from AML cells of a 71-year-old male Acute Myeloid Leukaemia patient with TP53 gene mutation (TP53: c.824G > A, p.Cys275Tyr) using episomal plasmids encoding the factors OCT4, SOX2, KLF4, L-MYC and human miR-302. The IPS-AML2-C3 (SYSUSHi002-A) iPSC line displayed typical embryonic stem cell-like morphology, carried the TP53 gene mutation, expressed several pluripotent stem cell makers, retained normal karyotype (46, XY), and was capable of forming three germ layer cells in vitro.

Structure-based identification of new orally bioavailable BRD9-PROTACs for treating acute myelocytic leukemia

BRD9 is essential in regulating gene transcription and chromatin remodeling, and blocking BRD9 profoundly affects the survival of AML cells. However, the inhibitors of BRD9 suffer from various drawbacks, including poor phenotype and selectivity, and BRD9 PROTACs still face the challenge of druggability, which limits the development of blocking BRD9 in AML. This study described an oral activity BRD9 PROTAC C6 by recruiting the highly efficient E3 ligase. C6 demonstrated remarkable efficacy and selectivity in BRD9 degradation with a BRD9 degradation DC50 value of 1.02 ± 0.52 nM and no degradation of BRD4 or BRD7. Moreover, our findings highlighted its therapeutic potential, as evidenced by profound in vitro activity against the AML cell line MV4-11. Furthermore, C6 exhibited superior oral activity, with a Cmax value of 3436.95 ng/mL. These findings demonstrated that C6, as a novel BRD9 PROTAC with remarkable pharmacodynamic and pharmacokinetic properties, had the potential to be developed as a promising therapeutic agent for AML treatment.

Regulatory role of RBM39 in acute myeloid leukemia: Mediation through the PI3K/AKT pathway

BackgroundAcute myeloid leukemia (AML) presents ongoing therapeutic challenges due to its intricate molecular pathogenesis. This study aimed to elucidate the role of RNA binding motif protein 39 (RBM39) in AML cell proliferation, apoptosis, and chemosensitivity, and its potential modulation of the PI3K/AKT pathway. MethodsIn vitro and in vivo experiments were conducted using AML cell lines (K562 and U937) and bone marrow mononuclear cells (BM-MNCs) from AML patients and healthy donors. RBM39 mRNA and protein levels were measured using qRT-PCR and Western blotting. Cells were transfected with sh-RBM39 or sh-control, and then treated with daunorubicin (DNR) or homoharringtonine (HHT) at varied concentrations. Cell proliferation, chemosensitivity, and apoptosis were assessed through CCK-8 assay and Annexin V–APC/PI staining. RNA sequencing identified differentially expressed genes (DEGs) post RBM39 knockdown. An in vivo xenograft AML model using E7070, a selective RBM39 inhibitor, was employed to evaluate RBM39 modulation effects. ResultsElevated RBM39 levels were found in AML patients and cell lines compared to controls. RBM39 knockdown promoted apoptosis, curtailed cell proliferation, and enhanced chemosensitivity to DNR and HHT in vitro. Drug-resistant or relapsed AML patients displayed higher RBM39 levels. RNA sequencing after RBM39 knockdown revealed downregulated PI3K/AKT signaling. The xenograft model validated in vitro results, as E7070 treatment suppressed AML xenograft growth via RBM39-mediated PI3K/AKT pathway suppression. ConclusionRBM39 plays a pivotal role in AML progression through the PI3K/AKT signaling pathway. Targeting RBM39, potentially with E7070, could inhibit proliferation and induce apoptosis in AML cells, offering a promising avenue for future AML research and treatment.

Cardiac glycoside ouabain efficiently targets leukemic stem cell apoptotic machinery independent of cell differentiation status

BackgroundAcute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)—the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored.MethodsDigoxin, a user-designed digitoxigenin-α-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation—one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery.ResultsPrimitive AML cells containing CD34+ LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation.ConclusionsOur data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery.53GxHmRcFt-KhVdMmSCJrsVideo

Abnormal adipogenic signaling in the bone marrow mesenchymal stem cells contributes to supportive microenvironment for leukemia development

BackgroundAcute myeloid leukemia (AML) is an aggressive hematological malignancy, associated with unfavorable patient outcome, primarily due to disease relapse. Mesenchymal stem cells (MSCs) residing in the bone marrow (BM) niche are the source of mesenchyma-derived subpopulations, including adipocytes, and osteocytes, that are critical for normal hematopoiesis. This study aimed to characterize BM-derived adipocyte/osteocyte fractions and their crosstalk with AML cells as a potential mechanism underlying leukemogenesis.MethodsBM cell subpopulations derived from primary AML patients were evaluated using humanized ex-vivo and in-vivo models, established for this study. The models comprised AML blasts, normal hematopoietic stem and progenitor cells and mesenchymal stromal subpopulations. ELISA, FACS analysis, colony forming unit assay, whole exome sequencing and real-time qPCR were employed to assess the differentiation capacity, genetic status, gene expression and function of these cell fractions. To explore communication pathways between AML cells and BM subpopulations, levels of signaling mediators, including cytokines and chemokines, were measured using the ProcartaPlex multiplex immunoassay.ResultsThe study revealed deficiencies in adipogenic/osteogenic differentiation of BM-MSCs derived from AML patients, with adipocytes directly promoting survival and clonogenicity of AML cells in-vitro. In whole exome sequencing of BM-MSC/stromal cells, the AHNAK2 gene, associated with the stimulation of adipocyte differentiation, was found to be mutated and significantly under-expressed, implying its abnormal function in AML. The evaluation of communication pathways between AML cells and BM subpopulations demonstrated pronounced alterations in the crosstalk between these cell fractions. This was reflected by significantly elevated levels of signaling mediators cytokines/chemokines, in AML-induced adipocytes/osteocytes compared to non-induced MSCs, indicating abnormal hematopoiesis. Furthermore, in-vivo experiments using a fully humanized 3D scaffold model, showed that AML-induced adipocytes were the dominant component of the tumor microenvironment, providing preferential support to leukemia cell survival and proliferation.ConclusionsThis study has disclosed direct contribution of impaired functional, genetic and molecular properties of AML patient-derived adipocytes to effective protection of AML blasts from apoptosis and to stimulation of their growth in vitro and in vivo, which overall leads to disease propagation and relapse. The detected AHNAK2 gene mutations in AML-MSCs point to their involvement in the mechanism underlying abnormal adipogenesis.AaFEVfq6u-1YqH7bnQ85uaVideo

Asymmetric anti-CLL-1×CD3 bispecific antibody, ABL602 2+1, with attenuated CD3 affinity endows potent antitumor activity but limited cytokine release

BackgroundAcute myeloid leukemia (AML) is a type of leukemia in adults with a high mortality rate and poor prognosis. Although targeted therapeutics, chemotherapy, and hematopoietic stem cell transplantation can improve...

Irradiated Extramedullary Acute Myeloid Leukemia Increases Survival in a Leukemic Mouse Model

The systemic nature of AML likely limits their immunogenicity because antigen presentation is primarily mediated by splenic dendritic cells, which induces immune tolerance. Since antigen presentation in tissue draining lymph nodes can induce anti-tumor immune responses, we hypothesized that local irradiation of cutaneous, extramedullary leukemias will increase immunogenic cell death, dendritic cell maturation and improved survival. A murine AML cell line C1498 was assessed for immunogenic markers calreticulin (CALR), HMGB1 and cGAS-STING pathway using fluorescence cytometry and qRT-PCR. Syngeneic mice were injected with C1498 subcutaneously to model extramedullary lesions and intravenously to model systemic leukemias. Cells and subcutaneous tumor were focally irradiated with 2 Gy or 8 Gy. Two-way ANOVA and Student's t-test were used for paired-wise comparisons. Survival was estimated by Kaplan-Meier plot and groups were compared using log-rank test. C1498 cells or tumors irradiated with 2 Gy or 8 Gy showed increased CALR and HMGB1 as well as (cGAS/Md21b1; 5-fold; p<0.001) and interferon alpha (Ifna; 3-fold; p<0.001) after 48h (18- and 4-fold, respectively; p<0.05) compared to not treated controls (NTC). Irradiated C1498 subcutaneous tumors displayed increased CD11b-positive cells (9-fold; p<0.001), CD11c (6-fold; p<0.001), and MHCII-positive CD11c cells (4-fold; p<0.01) in irradiated tumors compared to NTC. Irradiation of subcutaneous C1498 tumors increased median survival in mice also injected with C1498 cells intravenously compared to non-irradiated mice bearing systemic leukemias (46 vs. 25 days; p<0.01). Irradiation of C1498 tumors combined with anti-PDL1 treatment further increased median survival in our leukemia model compared to untreated controls (>50 vs. <30 days; p<0.001). Irradiated extramedullary AML increased immunogenic markers and activation of immune effector cells that correlated with increased survival in mice also bearing systemic leukemia. These results suggest focal irradiation of extramedullary AML may facilitate eradiation of systemic leukemias.

Inhibition of PLK4 remodels histone methylation and activates the immune response via the cGAS-STING pathway in TP53-mutated AML

AbstractAcute myeloid leukemia (AML) with TP53 mutation is one of the most lethal cancers and portends an extremely poor prognosis. Based on in silico analyses of druggable genes and differential gene expression in TP53-mutated AML, we identified pololike kinase 4 (PLK4) as a novel therapeutic target and examined its expression, regulation, pathogenetic mechanisms, and therapeutic potential in TP53-mutated AML. PLK4 expression was suppressed by activated p53 signaling in TP53 wild-type AML and was increased in TP53-mutated AML cell lines and primary samples. Short-term PLK4 inhibition induced DNA damage and apoptosis in TP53 wild-type AML. Prolonged PLK4 inhibition suppressed the growth of TP53-mutated AML and was associated with DNA damage, apoptosis, senescence, polyploidy, and defective cytokinesis. A hitherto undescribed PLK4/PRMT5/EZH2/H3K27me3 axis was demonstrated in both TP53 wild-type and mutated AML, resulting in histone modification through PLK4-induced PRMT5 phosphorylation. In TP53-mutated AML, combined effects of histone modification and polyploidy activated the cGAS-STING pathway, leading to secretion of cytokines and chemokines and activation of macrophages and T cells upon coculture with AML cells. In vivo, PLK4 inhibition also induced cytokine and chemokine expression in mouse recipients, and its combination with anti-CD47 antibody, which inhibited the “don’t-eat-me” signal in macrophages, synergistically reduced leukemic burden and prolonged animal survival. The study shed important light on the pathogenetic role of PLK4 and might lead to novel therapeutic strategies in TP53-mutated AML.

Super Enhancer Regulatory Gene FYB1 Promotes the Progression of T Cell Acute Lymphoblastic Leukemia by Activating IGLL1.

Arising from T progenitor cells, T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignant tumor, accounting for 15% of childhood ALL and 25% of adult ALL. Composing of putative enhancers in close genomic proximity, super enhancer (SE) is critical for cell identity and the pathogenesis of multiple cancers. Belonging to the cytosolute linker protein group, FYB1 is essential for TCR signaling and extensively studied in terms of tumor pathogenesis and metastasis. Dissecting the role of FYN binding protein 1 (FYB1) in T-ALL holds the potential to improve the treatment outcome and prognosis of T-ALL. In this study, SEs were explored using public H3K27ac ChIP-seq data derived from T-ALL cell lines, AML cell lines and hematopoietic stem and progenitor cells (HSPCs). Downstream target of FYB1 gene was identified by RNA-seq. Effects of shRNA-mediated downregulation of FYB1 and immunoglobulin lambda-like polypeptide 1 (IGLL1) on self-renewal of T-ALL cells were evaluated in vitro and/or in vivo. As an SE-driven gene, overexpression of FYB1 was observed in T-ALL, according to the Cancer Cell Line Encyclopedia database. In vitro, knocking down FYB1 led to comprised growth and enhanced apoptosis of T-ALL cells. In vivo, downregulation of FYB1 significantly decreased the disease burden by suppressing tumor growth and improved survival rate. Knocking down FYB1 resulted in significantly decreased expression of IGLL1 that was also an SE-driven gene in T-ALL. As a downstream target of FYB1, IGLL1 exerted similar role as FYB1 in inhibiting growth of T-ALL cells. Our results suggested that FYB1 gene played important role in regulating self-renewal of T-ALL cells by activating IGLL1, representing a promising therapeutic target for T-ALL patients.

Predicting and overcoming resistance to CDK9 inhibitors for cancer therapy.

Abnormally activated CDK9 participates in the super-enhancer mediated transcription of short-lived proteins required for cancer cell survival. Targeting CDK9 has shown potent anti-tumor activity in clinical trials among different cancers. However, the study and knowledge on drug resistance to CDK9 inhibitors are very limited. In this study, we established an AML cell line with acquired resistance to a highly selective CDK9 inhibitor BAY1251152. Through genomic sequencing, we identified in the kinase domain of CDK9 a mutation L156F, which is also a coding SNP in the CDK9 gene. By knocking in L156F into cancer cells using CRISPR/Cas9, we found that single CDK9 L156F could drive the resistance to CDK9 inhibitors, not only ATP competitive inhibitor but also PROTAC degrader. Mechanistically, CDK9 L156F disrupts the binding with inhibitors due to steric hindrance, further, the mutation affects the thermal stability and catalytic activity of CDK9 protein. To overcome the drug resistance mediated by the CDK9-L156F mutation, we discovered a compound, IHMT-CDK9-36 which showed potent inhibition activity both for CDK9 WT and L156F mutant. Together, we report a novel resistance mechanism for CDK9 inhibitors and provide a novel chemical scaffold for the future development of CDK9 inhibitors.