Antileukemic Activity Research Articles

Venetoclax Plus High-Dose Cytarabine and Mitoxantrone (HAM-Ven) As Salvage Treatment for Relapsed/Refractory AML: Updated Results of the Phase-I/II SAL Relax Trial

Background Primary refractory disease and relapse remain the main obstacles for cure in AML. In fit patients, several salvage cytostatic options based on higher doses of cytarabine have been published, with complete remission (CR) rates ranging from 40-55%. The BCL2 inhibitor venetoclax has demonstrated impressive antileukemic activity when combined with hypomethylating agents or low-dose cytarabine in newly diagnosed unfit AML patients. In the phase-I part of the RELAX trial of the Study Alliance Leukemia (SAL), we demonstrated the feasibility of combining venetoclax with higher-dose cytarabine plus mitoxantrone (HAM) in relapsed AML, resulting in a CR/CRi rate of 92%. Here, we report the updated results from the phase-I and additional patients from the phase-II dose expansion in relapsed or refractory (r/r) AML patients. Methods Patients with r/r AML fit for intensive salvage treatment were eligible. Based on the safety results from the phase-I part, patients were treated with the recommended phase-II dose combination of 400 mg venetoclax PO QD (d1-14) including a 2-day ramp-up, followed by cytarabine 1000 mg/m 2 IV BID (d3-5) in combination with mitoxantrone 10 mg/m 2 IV QD (d5-7). Concomitant CYP3A inhibitors were allowed with the appropriate dose adjustments, patients did not receive G-CSF. Patients achieving a CR could enter a maintenance phase with 400 mg venetoclax PO QD over twelve 28-day cycles. This research was supported by a grant from AbbVie Inc. The trial is registered under NCT04330820. Results Between April 2020 and June 2023, 12 patients were enrolled in the dose-escalation part and 40 patients in the expansion part, of whom 38 were evaluable for response by the time of writing. Median follow-up was 6.7 months. Among the 12 patients in the dose-escalation part, 3 received 200 mg/m 2 cytarabine QD over seven days, 3 received 500 mg/m 2 BID over three days and 3 received 1000 mg/m 2 BID over three days, each with identical doses of venetoclax (400 mg PO QD d1-14) and mitoxantrone (10 mg/m 2 IV QD d5-7). Of all 38 evaluable patients, 17 (44.7%) had adverse genetic risk according to ELN2022, and 1 (2.6%) had progressed from antecedent MDS. Median age was 56 years (range, 26-74 years). All patients experienced CTCAE grade ≥3 cytopenias. A median number of two non-hematologic grade 3 and 4 adverse events (AEs) in 32 patients with sufficient data were recorded. Grade 3 and 4 AEs occurring in ≥ 10% of patients included febrile neutropenia, pneumonia, sepsis and gastrointestinal disorders. The 30- and 60-day mortality was 2.6% and 5.2%. Remission assessment showed CR/CRi in 31/38 patients (81.6%). Among the seven patients with no CR/CRi, six showed refractory disease and one died in aplasia before response assessment. CR/CRi rate was 100% (8/8), 81,8% (9/11) and 70.6% (12/17) in patients with favorable, intermediate, and adverse risk, respectively. Utilizing LAIP-based DFN MRD approach, 7/32 (21.9%) evaluable patients were classified as MRD negative (CR/CRi MRD-). In 7/25 (28%) CR/CRi MRD+ patients, the MRD burden was below 0.1% CD45 + events. Of all 31 CR/CRi patients, 26 are in ongoing CR/CRi, 16 received allogeneic HCT as postremission treatment. Three patients died in CR from infectious complications after allogeneic HCT or post-remission treatment and two relapsed and succumbed to progressive disease. Six patients commenced maintenance treatment, of whom four are in ongoing remission. Discussion The combination of venetoclax with higher doses of cytarabine and mitoxantrone (HAM-Ven) is a well-tolerated and very efficacious treatment option leading to complete remissions in 81.6% of patients with r/r AML studied in the RELAX trial. Safety and efficacy compare favorably with published results from the FLAG-Ida-Ven regimen, thereby providing a less dose-intense, fludarabin-free approach for the treatment of fit r/r AML patients. Enrollment into the RELAX trial is currently ongoing. Updated results will be presented at the meeting.

IL1RAP-Specific T Cell Engager (TCE) Antibody Efficiently Depletes Acute Myeloid Leukemia (AML) Leukemic Stem Cells (LSCs)

LSCs are self-renewal primitive cells that initiate and maintain AML. Because they are highly therapy resistant, they are often responsible for treatment failures. Due to the lack of a specific antigen (Ag) and their quiescent nature, LSCs have been challenging to target and eliminate. IL1RAP is highly expressed on most human AML cell lines and primary blasts, including LSC-enriched CD34+CD38- cells, but not on normal hematopoietic stem cells (HSCs). To leverage this observation, we generated over 50 unique mouse IL1RAP monoclonal (m) antibodies (Abs), grouped them based on sequence, and selected representative members from the clades to generate murine-human chimeric mAbs. One of the mAbs, termed #24, had the strongest antibody-dependent cell-mediated cytotoxicity (ADCC) properties and the greatest in vivo anti-AML activity. Thus, it was advanced to generate a bispecific (Bs) anti-IL1RAP/CD3 TCE Ab termed BiF002, which incorporates a human IgG1 Fc mutated to lack FcR binding. Incubation of BIF002 with healthy donor T cells and AML cell lines or primary AML blasts induced IL1RAP dependent T cell activation, and Ab-dependent T cell lysis of leukemic cells in a dose-, time- and effector to target (E: T) ratio-dependent manner. The IC 50 against MOLM13 and THP-1 was subnanomolar (0.047nM, 95%CI:0.036-0.060nM and 0.025nM, 95%CI:0.14-0.40nM, respectively), at E:T ratio 5:1 at 48 hours using resting T cells. The IC 50 of IL1RAP pos AML bulk and CD34+ blasts (n=6) was also subnanomolar (0.45nM; 95%CI: 0.28-1.96nM). Co-cultures of normal CD34+ cells with BIF002 and T cells did not activate T cells or eliminate the normal CD34+ cells. To test the antileukemic activity of BIF002 in vivo, we engrafted luciferase (luc)-expressing MOLM13 cells into NSG-SGM3 (NSGS) mice. On day (D) 3, in vitro expanded healthy donors' T cells (3x10 6/mouse, weekly), with either BIF002 or a control IgG (10µg,) were administered intravenously (IV) every 3 days x 4-weeks. Mice treated with T cells + BIF002 exhibited a 100-fold greater tumor reduction on D33 than other groups, as measured by bioluminescence imaging (BLI), and had a significantly longer overall survival (OS; median 54.5 D) compared to IgG (27 D, p=0.0045), BIF002 alone (36 D, p=0.0049), or T cells + IgG (33 D, p=0.0062) treated controls. In initial dose finding studies using a similar schema, lower BIF002 doses (0.1 and 1 µg) were tested. Mice treated with T cells and 1 µg of BIF002 had a 100-fold tumor reduction on D29 and longer OS (median 57 D) compared to T cells + 0.1µg of BIF002 (31 D, p=0.0018) or T cells + vehicle (34 D, p=0.0019). We also engrafted luc-expressing primary blasts from a relapsed/refractory complex karyotype AML patient into NSGS mice that treated for 3 weeks using the above described schema (10µg). In this experiment, we used as control BIF026, a bispecific Ab (BsAb) carrying point mutations in both Fab arms to abrogate target binding to IL1RAP and CD3. Mice treated with T cells + BIF002 exhibited a >1000-fold reduction in disease burden and longer OS than BIF026 or BIF002 alone or T-cells+ BIF026-treated mice (median 46 D vs 26, 25 or 30 D, respectively, all comparisons, p=0.0003). In a second patient-derived xenograft (PDX) model, blasts from a relapsed complex karyotype AML patient were engrafted into NSG mice. Treatment was initiated on D10, with T cells given weekly along with 10µg of BIF002 or BIF026 for 3 weeks. After 70 days, all mice treated with T cells + BIF002 were alive, with no detectable blasts in peripheral blood or bone marrow (BM), while all vehicle (median 38 D, p=0.0002) and T cells + BIF026 (38 D, p=0.0001) treated controls had died of leukemia. To determine if T cells + BIF002 eradicated LSCs, we randomly selected 3 female mice from each group on D 34 and transplanted BM mononuclear cells (MNC) into secondary NSGS recipients (n=7). At D 90, no recipients of BM from donors treated with T cells + BIF002 had detectable blasts and remained alive, while all vehicle (median 26 D, p=0.0004) and T cells + BIF026 (median 26 D, p=0.0002) treated controls succumbed to AML. Importantly, no obvious hematopoietic or non-hematologic side effects were observed after BIF002 administration alone or with activated T cells in normal and leukemic mice. In summary, we provide evidence of the subnanomolar potency and efficacy of BIF002, a novel anti-IL1RAP/CD3 TCE, in eliminating AML blasts, including LSCs. BIF002 is currently undergoing IND-enabling studies.

Naked Antibody-Based Conditioning Targeting CD47 and CD117 Plus Janus Kinase Inhibition Enables Toxin-Free, Radiation-Free, and Chemotherapy-Free Fully Mismatched Allogeneic Hematopoietic Stem Cell Transplantation

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for numerous malignant and non-malignant hematologic diseases. To enable engraftment of donor hematopoietic stem cells (HSCs), recipients undergo conditioning with chemotherapy and irradiation to make marrow space for incoming donor HSCs and suppress host immune responses that may lead to graft rejection. These cytotoxic conditioning regimens carry risks of severe toxicities which prevents many patients from accessing the lifesaving benefit of allo-HSCT. Previously, we developed fully ablative CD45- and cKit-targeted antibody-drug conjugates (ADC) which, when combined with Janus kinase 1/2 (JAK1/2) inhibitors, enabled fully mismatched allo-HSCT with full donor chimerism and minimal toxicity while providing durable antileukemia activity (Persaud et al (2022), ASH Abstract). However, even ADCs carry potential risks of acute and chronic toxicities that may preclude their common use as HSCT conditioning agents. Moreover, unlike malignant diseases, allo-HSCT for non-malignant diseases does not require the conditioning regimen to provide antitumor benefit and partial donor chimerism may be sufficient for cure, strongly favoring the use of less intense conditioning strategies with the fewest possible toxicities. We hypothesized that naked antibody-based conditioning strategies can achieve the therapeutic benefits of allo-HSCT for non-malignant hematologic diseases, obviating exposure to toxic ADC payloads. METHODS: We combined unconjugated antibodies targeting CD47 and cKit with JAK1/2 inhibition to perform minor histocompatibility antigen (miHA)-mismatched (129/Sv→B6-Ly5.1) and fully mismatched (BALB/c→B6) murine allo-HSCT. In some full mismatch HSCT experiments, recipients included B6-gp91phox -/- mice, which serve as a model of chronic granulomatous disease (CGD) due to their deficiency in a catalytic subunit of NADPH oxidase. Mice received 100 µg anti-CD47 (clone mIAP410; BioXcell) 8 days before HSCT (d-8) as a priming dose to reduce the severity of CD47 blockade-induced anemia. Next, mice received 500 µg anti-cKit (clone ACK2; BioLegend) on d-6 and daily 500 µg doses of anti-CD47 from d-6 through d-2. For JAK1/2 inhibition, mice received oral ruxolitinib (Incyte; 2 mg drug/g chow) starting d-3 or d-1 and continuing until d+28. On d0, recipients were transplanted with 20 x 10 6 whole bone marrow cells from 129/Sv (miHA) or BALB/c (fully mismatched) mice. Peripheral blood donor chimerism was followed monthly by flow cytometry for CD45.2 + (miHA) or H-2D d+ (fully mismatched) cells. Oxidative burst among granulocytes in B6-gp91phox -/- recipients was assessed by dihydrorhodamine 123 (DHR123) staining after phorbol 12-myristate 13-acetate (PMA) stimulation. RESULTS: CD47 and cKit antibodies plus ruxolitinib enabled multilineage engraftment in both miHA and fully mismatched allo-HSCT, including myeloid lineage donor chimerism of 95-99% (Figure 1A). As expected, CD47 blockade induced significant but transient morbidity from anemia during the peritransplant period; notably, a shorter duration of ruxolitinib treatment before HSCT (starting d-1 instead of d-3) improved the tolerability of the regimen without impacting engraftment success. Allo-HSCT of B6-gp91phox -/- mice led to near complete replacement of host granulocytes in peripheral blood with donor granulocytes, whose DHR123 positivity upon PMA stimulation indicated an intact oxidative burst capacity (Figure 1B). CONCLUSIONS: Combination of antibodies targeting CD47 and cKit with JAK1/2 inhibition provides a novel conditioning strategy for allo-HSCT in mouse models, achieving high-level, stable multilineage engraftment without radiation, chemotherapy, or exposure to toxic ADC payloads. The near complete donor chimerism among granulocytes made it particularly well suited to correcting phagocyte oxidase function in the gp91phox -/- model of CGD. Future studies seek to 1) mitigate the peritransplant anemia caused by CD47 blockade using Fc-silenced anti-CD47 reagents, 2) determine the proportions of donor- versus recipient-derived myeloid cells in non-hematopoietic tissues of allo-HSCT recipients, and 3) use murine infection models to evaluate the impact of allo-HSCT on host defense in transplanted B6-gp91phox -/- recipients.

CD19-CAR T Cytotoxicity Is Improved By AMPKγ2 Overexpression, Which Is Further Enhanced By Metformin Treatment

Background Acute lymphoblastic leukemia (ALL) is the most common leukemia found in children. Chimeric Antigen Receptor (CAR) T cells induce high response rates against relapsed/refractory B-cell ALL, but 40% of CAR-T recipients suffer disease relapse, many due to poor in vivo survival of the CAR-T cells. Metabolic exhaustion has been proposed as a major barrier to optimal CAR-T cell performance. Therefore, modulating T cell metabolism may represent a promising method to improve CAR T-cell therapy. AMP-activated protein kinase (AMPK) is a heterotrimeric signaling complex which serves as a cellular energy sensor, promoting mitochondrial health and oxidative metabolism under conditions of energetic stress. We have demonstrated overexpressing the regulatory subunit AMPKγ2 increases AMPK signaling in human T cells, and hypothesized that this overexpression would enhance CAR-T metabolic fitness and improve anti-leukemia activity. Results We used lentiviral transduction to introduce a CD19-reactive, CD28 CAR into human T cells, followed by transduction of either AMPKγ2 or an empty vector (EV) control. Dual transduced cells were isolated by flow sorting and cultured with human IL2. The metabolism and cytotoxicity of these CAR-T cells were first assessed in vitro. AMPK-CART cells showed higher oxidative metabolism, with a 29% increase in basal oxygen consumption rates (OCR) and a 45% increase in maximal OCR (p<0.001) following overnight stimulation by NALM6 cells. In addition, co-culture with Zs-Green+ NALM6 cells revealed greater cytotoxicity by AMPK-CART cells (p<0.001) (Fig. 1A), accompanied by a 35% increase in CD25 expression (p<0.05). We then pivoted to studying AMPK-CART cells in vivo utilizing a murine xenograft leukemia model. Interestingly, despite their in vitro advantages, AMPK-CART cells demonstrated equivalent anti-tumor capacity in vivo. We reasoned that AMPKγ2 overexpression may simply protect AMPK signaling which is already occurring. To test this idea, we coupled AMPKγ2 overexpression with transient metformin treatment, a process known to activate AMPK signaling through inhibition of mitochondrial complex I. Metformin treatment was followed by the functional analysis of CAR-T cells in a chronic stimulation protocol where we repetitively stimulate CAR-T cells with NALM6 targets to induce exhaustion. After 8 days of chronic stimulation, metformin treated AMPK-CART cells (7mM for 4 hours) demonstrated the highest cytotoxicity against NALM6 cells compared to either metformin treated EV-CART cells or AMPK-CART cells without metformin treatment (Fig. 1B). This cytotoxic advantage was accompanied by an 18% increase in CD25 expression (2052.67±104.74 vs. 1742.67±14.64, p<0.05) and a 56% elevation in the percentage of central memory cells (9.15±1.32 vs. 5.88±0.25, p<0.05) in metformin treated AMPK-CART cells. These changes were not seen in EV-CART cells with metformin treatment. Conclusions Here, we report that AMPKγ2 overexpression in CD19-CAR T cells enhances oxidative metabolism and improves in vitro cytotoxicity but does not significantly increase in vivo anti-leukemia function. Metformin pre-treatment elevates CAR-T cytotoxicity following chronic antigen stimulation, but only when coupled with AMPKγ2 overexpression. This combination is what we predict will improve the function of CD19-CAR T cells in our murine xenograft leukemia model and human samples.

Anti-Leukemic Activity of Luveltamab Tazevibulin (LT, STRO-002), a Novel Folate Receptor-α (FR-α)-Targeting Antibody Drug Conjugate (ADC) in Relapsed/Refractory CBF2AT3::GLIS2 AML

Introduction: CBFA2T3::GLIS2 (CBF-GLIS) is an oncogenic fusion associated with a lethal and highly refractory childhood myeloid leukemia manifested by the RAM phenotype, uniquely expressed in infants and young children. Conventional chemotherapy and stem cell transplant (SCT) result in extraordinarily high rates of induction failure (>80%) and near uniform fatality with 2-year survival <15%. When conventional therapy fails, this leukemia is refractory to all reinduction therapies with post-relapse survival of nearly 0%. We have previously reported that FR-α is expressed on the surface of CBF-GLIS AML cells and that LT, an investigational ADC, demonstrated potent in vitro and in vivo anti-leukemia activity in CBF-GLIS AML cell lines and xenograft models (PMID: 36149945). LT has been provided under single patient IND to pts with CBF-GLIS AML. Herein, we update the clinical experience. Methods: All pts were required to demonstrate CBF-GLIS fusion by PCR or sequencing. Each investigator in consultation with Sutro Biopharma Inc. defined the treatment plan as to dose, schedule, and combination therapy administered. Results: Between August 2021 and March 2023, 25 pts with relapsed/refractory CBF-GLIS AML were treated with LT at doses of 4.3 or 5.2 mg/kg every 2-4 weeks (4 received smaller fractionated doses (FD) on days 1, 3, 5 per cycle). Median age at treatment was 2 years (range 0.7-8) with median number of prior therapies of 2 (range 1-7). 10 pts had relapsed after SCT. The remaining 15 had primary refractory, relapsed or MRD+ AML. Median time from initial diagnosis was 7.9 months (range 1.5-82.5). Of the 25 pts who received LT, 18 had ≥5% blasts (morphologic disease, MD) and 7 had <5% blasts (sub-morphologic disease, SMD). 21 pts initially received LT monotherapy (mono) (15 with MD and 6 with SMD), 9 of which went on to receive combination (combo) with additional therapies. The remaining 4 received LT in combo upfront. Morphologic or molecular/MRD- response was observed in 12/25 pts (48%). Among those with MD, 7/18 (39%) reached a CR/CRh (5 MRD-) and among those with SMD, 5/7 (71%) reached MRD- CR. Median number of doses received before response was 2. Responses were observed among patients receiving both mono and combo. Among the 11 MD pts treated with mono (excluding FD), 3 reached CR (27%). Among 10 pts with MD who received combo either initially or following mono, 4 reached CR/CRh (40%). Among the SMD pts, 4 reached MRD- with monotherapy and 1 with combo. Of the 10 pts with post-SCT MD relapse, 7 patients received non-fractionated LT either as mono or combo therapy. Of these, the CR rate was 71% (5/7). LT therapy enabled these children to receive potentially definitive therapy. Of the 7 pts with MD in CR, 4 proceeded to SCT, 2 received donor lymphocyte infusions and 1 received consolidation therapy. Of the 5 pts with SMD in MRD- CR, 1 proceeded to SCT, 2 received consolidation therapy in preparation for SCT, and 2 received LT maintenance therapy. In total, 12 pts remain on treatment. Of the 25 pts who received LT, 17 (63%) are alive, 7 died of progressive disease (5 with relapse post-SCT) and 1 died from SCT-related complications. Of the 10 pts who received LT (non-fractionated) post-transplant, 7 are alive (78%). Pt status and treatment are shown in Figure 1. LT was generally well tolerated. TEAEs reported at an incidence of ≥ 25% included decreased neutrophil, platelet, WBC, or lymphocyte count, anemia, increased AST or ALT, diarrhea, and pyrexia. Grade 3+ TEAEs (≥ 10%) included decreased neutrophil, platelet, WBC, or lymphocyte count, anemia, febrile neutropenia, ALT increased, vomiting and hypoxia. Observed cytopenias were often reported in pts with baseline cytopenia. Grade 5 SAEs were limited to fractionated dosing pts: apnea (n=1) and cardiopulmonary failure (n=1). Neither event was considered related to LT. The median duration of LT treatment was 9.1 weeks (2.1-60) and 60% of pts received ≥5 doses. While the initial dose was typically delivered inpatient, approximately 80% of subsequent doses were delivered outpatient. Conclusion: LT based therapy in CBF-GLIS AML provides clinical and laboratory responses that are highly unusual in this refractory patient population. LT has an acceptable safety profile and does not require inpatient delivery. Importantly, LT appears to be enabling these children to receive potential definitive therapy. These data support further characterization of this drug in pediatric CBF/GLIS AML.

Maintenance Therapy with Chidamide Plus Azacitidine after Allogeneic Hematopoietic Stem Cell Transplantation for High-Risk Acute Myeloid Leukemia Patients

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is theoretically the only curative option for high-risk acute myeloid leukemia (AML) patients. However, many patients relapse after allo-HSCT, and treatment options are very limited. Azacitidine, a DNA methyltransferase inhibitor exhibits anti-leukemia activity in myeloid diseases. Chidamide, a selective histone deacetylase inhibitor, effectively induces apoptosis of AML cells. They have synergistic inhibitory effect on AML cells when combined together. Therefore, we conducted a multicentre, prospective study (ChiCTR2300067593) to examine the tolerability and efficacy of a combination of both chidamide and azacitidine as a maintenance therapy for high-risk AML patients after allo-HSCT. Patients and methods： Patients ≤ 60 years of age with high-risk AML post- allo-HSCT were enrolled. The high-risk characteristics of AML were defined as one or more of the following criteria: a) Patients with poor prognosis according to the genetic risk stratification assessment of European Leukmia Net (ELN) in 2022; b) Primary refractory or recurrent AML; c) Secondary AML (secondary to MDS or treatment-related AML). All patients received Chidamide 5 mg/d combined with azacitidine 75 mg/m2/d for 5 days. The cycle interval was 6-8 weeks. A total of 6 cycles was recommended. Treatment started as early as 3 months after transplantation. The primary endpoint of this study was cumulative incidence of relapse (CIR). The secondary endpoints included rates of overall survival (OS) and event-free survival (EFS). Survival outcomes were estimated using Kaplan-Meier analysis. Results： Thirty-six patients have been recruited. The median age was 42 years (range, 16-59). Baseline characteristics are shown in Table 1. With a median follow-up time of 412（185-762）days, the 1-year CIR was 12.3 (6.5-18.1) %, the 1-year OS and EFS rates were 100.0% and 87.7 (81.9-93.5) %, respectively. The median OS and EFS time were not reached. Four patients relapsed, two died, and two are still alive and ready for the second transplantation. The most common adverse events (AEs) were thrombocytopenia reaction and gastrointestinal tract reaction. Grade 2 or 3 AEs were observed in 16.7% (6/36) and 25.0% (9/36) of the patients, respectively. No grade >3 AEs were noted. 9 patients (6/36,16.7%) developed grade 3-4 thrombocytopenia, without active hemorrhage. All of them received blood transfusion and thrombopoietin receptor agonist. The median persistence of thrombocytopenia was 3 (2-5) d. Chronic graft-versus-host-disease (cGVHD) occurred in 25.0% (9/36) of patients. Four cases (4/36, 11.1%) were complicated with infection. They were one case of intestinal infection, one case of pulmonary and EBV infections, one case of hepatitis B virus activation and one case of herpes zoster. And all infected patients recovered safely after anti-infection and symptom-oriented treatment. CMV activation was not observed. Non relapse mortality (NRM) was zero. Conclusions： We conclude that chidamide plus azacitidine can be administered safely after allo- HSCT with no evidence of an increased incidence of GVHD, and this combination may decrease the relapse rate in high-risk AML patients. This novel maintenance therapy may be a promising way to prevent relapse in high-risk AML patients.

Epigenetic Reprogramming By Histone Deacetylase Inhibitors Improves CAR-T Cell Functions

Background: The exhaustion and diminished persistence of CAR-T cells primarily affect clinical efficacy and long-term outcomes. Epigenetic is an innate mechanism in regulating the biological function CAR-T cells. We previously dissected comprehensive dynamic changes of chromatin accessibility and transcription factors during CAR-T cell exhaustion (Jiang P, et al. Leukemia. 2022 Nov;36(11):2656-2668). Therefore, combined application with epigenetic drugs have much therapeutic potential. Methods: We initially performed an extensive screening among 370 epigenetic drugs to select candidates that could resist exhaustion and maintain phenotype of memory subsets, and thoroughly verify their effects on CAR-T cells in vitro. Next, we conducted multi-omics analysis comprising RNA-seq, ATAC-seq, and CUT&Tag-seq to explore underlying regulative mechanisms of epigenetic manipulation by epi-drugs. In the end, we used preclinical B-ALL NSG mouse model to verify anti-tumor activities of drug-treated CAR-T cells in vivo. Results: Among those screened epigenetic drugs, we identified two histone deacetylase inhibitors (HDACi), Chidamide and M344, could best promote maintenance of central memory subsets and resistance to exhaustion of CAR-T cells, with enhanced anti-leukemia activities in vitro (Figure 1a-c). Chidamide and M344 inhibited enzymatic activity and expression of HDAC1, which elevated the acetylation level of histone H3, particularly at H3K27 in CD19 CAR-T cells. Genetic knock-down of HDAC1 could partially reflect the phenotypic mimicry by Chidamide and M344 on CAR-T cells. By combining multi-omics data from bulk RNA-seq, ATAC-seq, and H3K27ac-targeted CUT&Tag-seq in Chidamide-or M344-treated CAR-T cells, we identified distinct patterns of epigenetic reprogramming, and enrichment of genes associated with exhaustion, as well as regulation of cellular differentiation and formation of memory subsets. In addition, analysis of motif and interaction network among transcription factors revealed altered regulation of gene transcription (data not shown). Finally, either Chidamide-pretreated CAR-T cells or in vivo administration of Chidamide after CAR-T cells infusion had exhibited enhanced and persistent tumor clearance in preclinical B-ALL NSG mouse model (Figure 1d). Conclusion: we conducted comprehensive screening of 370 epigenetic drugs and identified that administration of HDACi, particularly Chidamide and M344, could induce significantly enhanced anti-tumor function with maintenance of memory phenotype and reduced exhaustion in CAR-T cells through epigenetic reprogramming and altered regulation of gene transcription, which provided with basis and therapeutic targets for the rational combination of CAR-T cell therapy and epigenetic drugs in patients with hematological malignancies. Figure legends: Figure1. Screening of 370 epigenetic drugs identified M344 and Chidamide that enhanced maintenance of memory phenotype, resistance to exhaustion, and ability of tumor clearance in CAR-T cells. (a) The scatterplot of screening 370 compounds based on relative CD45RO +CD62L + proportion of central memory in treated CAR-T cell, showing of compared with DMSO control group by flow cytometry. Red indicates an increase in the proportion of memory CAR-T cells, while blue represents a decrease. (b) Frequency of co-expression of exhaustion-related inhibitory receptors (LAG-3, TIM-3, and PD-1) in DMSO or HDACi-treated groups. Data presented as mean ± SEM of independent donors (n=3 or more). (c) Flow cytometric analysis of central memory and effector memory subsets in CAR-T cells in each group. Data presented as mean ± SEM of independent donors (n=3 or more). (d) Bioluminescent imaging of tumor clearance in B-ALL NSG mouse. ‘X’ represents dead subject (end point). Survival of B-ALL NSG mice i.v. infused with T cells or treated CAR-T cells. Statistical analysis by Mantel-Cox test. Data presented as pooled from independent experiments (n=2).

The anti-leukemia activity and mechanisms of shikonin: a mini review.

Leukemia encompasses a group of highly heterogeneous diseases that pose a serious threat to human health. The long-term outcome of patients with leukemia still needs to be improved and new effective therapeutic strategies continue to be an unmet clinical need. Shikonin (SHK) is a naphthoquinone derivative that shows multiple biological function includes anti-tumor, anti-inflammatory, and anti-allergic effects. Numerous studies have reported the anti-leukemia activity of SHK during the last 3decades and there are studies showing that SHK is particularly effective towards various leukemia cells compared to solid tumors. In this review, we will discuss the anti-leukemia effect of SHK and summarize the underlying mechanisms. Therefore, SHK may be a promising agent to be developed as an anti-leukemia drug.

Pivekimab Sunirine (PVEK, IMGN632), a CD123-Targeting Antibody-Drug Conjugate, in Combination with Azacitidine and Venetoclax in Patients with Newly Diagnosed Acute Myeloid Leukemia

Background In unfit patients (pts) with newly diagnosed (ND) AML, long-term survival remains short (mOS 14.7 months) despite improved responses (CR 37% and CR/CRi 66%) with azacitidine (AZA) and venetoclax (VEN) (VIALE-A, DiNardo NEJM 2020). In a pooled analysis of AZA-VEN in ND AML pts with poor risk cytogenetics the response rates were higher in TP53wt pts (CR/CRi 70%) compared with TP53mut pts (CR/CRi 41%) (Pollyea Clin Cancer Res 2022). The measurable residual disease (MRD)-negative rate was 41% in AZA-VEN treated pts in VIALE-A which was associated with improved survival (Pratz JCO 2022). Pivekimab sunirine (PVEK, IMGN632) is a first-in-class antibody-drug conjugate (ADC) comprising a high-affinity CD123 antibody, cleavable linker, and an indolinobenzodiazepine pseudodimer (IGN) payload. The novel IGN payload alkylates DNA and causes single strand breaks without crosslinking (Kovtun Blood Adv 2018). Methods This is an ongoing, open-label, multicenter, Phase 1b/2 study of PVEK in combination with AZA +VEN in adults with ND CD123-positive AML (any CD123+ expression by local flow cytometry or IHC). Pts received the recommended phase 2 dose of PVEK 0.045 mg/kg IV on D7 + AZA 75 mg/m 2 SC or IV daily on D1-7 + VEN up to 400 mg PO daily for 14 to 28 days (based on cohort assignment) in a 28-day cycle. Bone marrow assessment was performed at D14 (cohort 1) or D21 (cohort 2) of cycle 1 to determine VEN duration. The primary endpoints are composite CR rate (CCR [CR+CRh+CRp+CRi]), MRD rate (assessed centrally [Hematologics, Inc.] by flow cytometry; <0.1% defined as negative) and duration of remission. Key secondary endpoints are safety, pharmacokinetics and immunogenicity. Responses were determined using ELN 2017 criteria (with the addition of CRh) and a 14-day post-marrow count recovery window. Results As of July 17, 2023, data is available for 50 ND pts (n=25 per cohort) treated with PVEK+AZA+VEN and are aggregately reported here. The median age was 74 years (range, 46-83), 42% were ≥ 75 years old, 26% had secondary AML, 64% were ELN 2017 adverse risk, and 38% had a TP53mut. The most common non-hematologic treatment-emergent adverse events (TEAE) (all grades [grade 3+]) seen in > 20% of all pts were febrile neutropenia (44% [38%]), constipation (42% [2%]), peripheral edema (36% [4%]), diarrhea (36% [2%]), hypophosphatemia (32% [0%], nausea (28% [4%]), and hypokalemia (24% [2%]). No veno-occlusive disease (VOD) events were reported. In responders, the median time to achieve absolute neutrophil count ≥500/µL and platelet count ≥50,000/µL was 35.5 days (range, 20-55) and 22 days (range, 20-55), respectively. The median post-remission cycle delay (beyond cycle day 28) was 14 days. Two pts discontinued PVEK due to a TEAE (edema; marrow aplasia). The 60-day mortality was 4%. In the ITT population (N=50) the CR rate was 52% (26/50), and the CCR rate was 66% (33/50). In pts ≥ 75 years old, the CCR rate was 76% (16/21). In pts known to be TP53wt, both the CR and CCR rate was 88% (22/25). Additional response subsets in Table 1. Twenty-one pts received 9-14 days of VEN, 9 pts received 15-21 days of VEN and 20 pts received ≥ 22 days of VEN in cycle 1; of those who received ≤ 14 days of VEN the CR rate was 71% (15/21) and the CCR rate was 76% (16/21). Of the 26 pts who achieved CCR and had an MRD-evaluable sample, 73% (19/26) achieved MRD negativity (CCR MRD-, <0.1% residual blasts); notably all had undetectable MRD levels (lower level of detection is 0.02%). MRD-negativity was achieved rapidly with a median of 1.7 months (range, 0.8-4.5). CCR MRD- rates were high across major molecular subsets, including FLT3 (ITD/TKD) at 100% (7/7), IDH1/2 at 89% (8/9), and NPM1 at 89% (8/9), as well as ELN risk groups (intermediate 80% [8/10]; adverse 64% [9/14]). Conclusion With a manageable safety profile in pts with ND AML, the PVEK+AZA+VEN triplet demonstrated anti-leukemia activity with robust CR rates and early MRD negative responses in a cohort enriched for adverse disease characteristics (64% ELN adverse risk; 38% TP53mut). Encouraging CCR MRD- rates were observed across cytogenetic/molecular subsets, and the majority of responding pts achieved early and deep remissions, which may translate to improved clinical outcomes. The regimen was well tolerated with no new safety signals, and the addition of PVEK to the AZA-VEN backbone did not appear to meaningfully prolong count recovery.

The Topoisomerase-I Inhibitor Gimatecan Exhibits Potent and Selective Activities Against B-Cell Precursor Acute Lymphoblastic Leukemia with a Favorable Cardiotoxicity Profile

Topoisomerase-II inhibitors such as daunorubicin have long been the standard chemotherapeutic agents for managing acute leukemias. Yet, severe complications could be resulted from their myelosuppressive and cardiotoxic nature. Here, we report preclinical evidence showing the exceptional activities of gimatecan, an orally available topoisomerase-I inhibitor currently being investigated in Phase II trials for solid cancers, on hematologic malignancies. Activity screening on leukemia cell lines revealed a substantially higher potency of gimatecan than frontline chemotherapeutics against B-cell precursor acute lymphoblastic leukemia (BCP-ALL) driven by on-target depletion of topoisomerase-I. Gimatecan had a median IC50 of 0.9 nM against BCP-ALL with profound selectivity over acute myeloid leukemia (AML, 3.6-fold, P< 0.01) or hematopoietic stem/progenitor cells (HSPCs, 75-fold, P< 0.001). In contrast, the four standard chemotherapeutics (cytarabine, daunorubicin, dexamethasone and vincristine) exhibited much higher median IC50s against BCP-ALL (18-2,201 nM). Following single-agent gimatecan treatment, complete dissolution of medullary and extramedullary leukemia (CNS and testis) by >95% inhibition concomitant with markedly extended survival (1.2 to 3.3-fold, P< 0.01) were demonstrated in animals grafted with cytogenetically distinct lymphoid leukemia cell lines (Reh, ETV6-RUNX1+; 697, TCF3-PBX1+; RS4;11, KMT2A-AFF1+; HAL-01, TCF3-HLF+) and in patient-derived xenografts from three cases of highly resistant BCP-ALL failing salvage chemotherapy or upfront immunotherapies. Splenocytes isolated from NSG mice relapsed from gimatecan treatment demonstrated recurrent drug sensitivity ex vivo comparing with vehicle controls and that gimatecan reinduction offered additional survival benefits in the xenograft model ( P< 0.05). The therapeutic efficacy was also observed at varied initial tumor load, together suggesting its potential for reinduction and flexibility on the administration schedule. In NSG mice reconstituted with cord blood HSPCs, gimatecan displayed a mild but transient suppression on normal hematopoiesis without bias on lineage output (63% reduction at 6 weeks, P=0.002; not statistically significant at 14 weeks). Importantly, gimatecan did not affect the viability of human iPSC-derived cardiomyocytes (hiPSC-CM) even at the highest tested dose (1,000 nM) as opposed to daunorubicin (IC50: 130 nM). Concordantly, no noticeable signs of DNA damage (by γ-H2AX staining) or apoptotic events (by enumeration of condensed nuclei) on hiPSC-CMs were observed after gimatecan exposure at doses at or above its Cmax (~130 nM). The gross structure of heart tissues harvested from gimatecan-treated mice was also intact without signs of cardiotoxicity at the dose effective to suppress leukemia (0.2 mg/kg). When compared with malignant B-lymphoblasts, there was a declined expression of topoisomerase-I in HSPCs (0.63-fold, P=0.025) and cardiomyocytes (0.24-fold, P<0.001), potentially explaining its selectivity against BCP-ALL. Mechanistically, gimatecan induced time- and dose-dependent cell death via intrinsic apoptosis coupled with G2/M cell cycle arrest. RNA-seq analysis of gimatecan-treated BCP-ALL cells showed activation of the tumor suppressive p53 pathway and suppression of the MYC pathway. Phosphorylation of p53 and appearance of DNA damage markers, including CHK1/2, PARP and H2AX, were robustly detected upon gimatecan treatment. In short, gimatecan possesses remarkable anti-leukemia activities and tolerable toxicity profiles compared to counterpart topoisomerase-II inhibitor. This study, therefore, puts forward a new agent for BCP-ALL and points towards randomized clinical trials to realize its potential on acute leukemias.

A Novel Natural Derivative GL-V9 Inducing Mitotic Catastrophe and Displaying a Synergistic Anti-Tumor Effect with EZH2 Inhibitor in Acute Myeloid Leukemia

Background GL-V9 is a flavonoid derivative from the natural product wogonin with elegant anti-tumor activity, while the effect of GL-V9 in acute leukemia has no report. Enhancer of zeste homolog 2 (EZH2) catalyzes trimethylation on the K27 residue of histone H3 (H3K27Me3), which facilitates non-homologous DNA end joining (NHEJ). EZH2 inhibitor could induce chromatin decondensation and promote the sensitivity of tumor cells towards DNA damage drugs. In the present study, we evaluated the anti-leukemia activity of GL-V9 and its synergy with EZH2 inhibitor DZNeP in acute myeloid leukemia (AML). Methods Cell proliferation and cytotoxicity were measured by Cell Counting Kit-8(CCK-8) in U937, THP-1, and MV4-11 AML cells. RNA-seq was performed in U937 cells treated with 2μM DZNeP, MV4-11 cells treated with 4μM GL-V9, and vehicle for 48 hours. The clone formation rate was measured by a plate clone formation assay. Cell cycle and apoptosis were measured by flow cytometry analysis. Morphology examination was conducted with Fluorescence (IF) microscopy . Expression of target genes was detected by western blot and RT-qPCR. Results GL-V9 had a dose-/time-dependent effect on cell viability with IC50 at 3-4μM in U937 and THP-1 cells (Fig.1A); it also significantly induced the G2/M arrest and apoptosis of the cells (Fig.1B, C). IF staining showed GL-V9 triggers micro-, multi-nucleation, and cytoskeleton abnormalities, the feature of mitotic catastrophe in U937 cells 4hrs after the treatment (Fig1.D). These data indicated that GL-V9 exerts the anti-leukemia effect by inducing mitotic catastrophe. We further analyzed the whole genome transcriptome upon GL-V9 treatment in U937 cells, and GSEA analysis of the differentially expressed genes (DEGs) showed that GL-V9 treatment negatively correlated with genes involved in sister chromatid separation, microtubule cytoskeleton organization, DNA double-strand break repair, mismatch repair, and cell cycle checkpoint signaling (Fig1.E). These data revealed that GL-V9 may induce DNA damage-mediated mitotic catastrophe in the AML cells. H3K27me3 and EZH2 play an important role in the early DNA damage response. We thus carried out the combination therapy of EZH2 inhibitor DZNeP with GL-V9. Results showed that co-treatment of GL-V9 with DZNeP exerted synergistic effects on reduced cell viability and clone formation versus single-drug in U937 and MV4-11 cells (Fig2.AB). DZNeP induced G0/G1 arrest; and GL-V9 induced G2/M arrest; whereas the combination significantly decreased the number of cells in the S-phase (Fig2.C). Also, the percentage of the apoptotic cells significantly increased upon the GL-V9+DZNeP treatment versus the controls in the cells (Fig2.D) as well as in ASXL1 mutant AML-M5 and AML-M2 primary cells (Fig2.E). Consistently, the combination significantly induced the expression changes of cell cycle regulator CCNE2 and p21, and dramatically up-regulated apoptotic effectors PARP, Caspase-3, and DNA damage marker γH2AX versus single-drug (Fig2.F). Furthermore, the overlapped DEGs were identified with 242 genes regulated in homo-direction but 25 regulated in the opposite treated with GL-V9 or DZNeP, these genes mainly influenced DNA replication, chromatin structures, and DNA damage response (Fig2.G). GL-V9 and/or DZNeP down-regulated the EZH2 expression, induced the decrease of p-AKT and c-Myc, and the increase of p53 in U937 cells (Fig. 2H). These data indicate that GL-V9+DZNeP may exert the anti-leukemia effect by inducing DNA damage via targeting EZH2/ PI3K/AKT signaling and the p53-c-Myc axis in the cells. EZH2 inhibition modifies the chromatin structure and decondensed the H3K27me3-marked chromatin, making AML cells more accessible to the nuclear-attacked agent GL-V9. The mechanism model is depicted in Fig. 2I. Conclusions GL-V9 is an effective novel nature compound against AML and exhibited strong synergistic effects with the epigenomic drug EZH2 inhibitor DZNeP potentially by inducing DNA damage-mediated mitotic catastrophe through targeting EZH2/ PI3K/AKT signaling and p53-cMyc axis. Our results provide pre-clinical evidence for the new combination as a promising clinical option for AML patients.

Enhancement of exercise expanded gamma-delta T-cells anti-leukemic activity with TIGIT blockade

Enhancement of exercise expanded gamma-delta T-cells anti-leukemic activity with TIGIT blockade

Selinexor Synergistically Promotes the Antileukemia Activity of Venetoclax in Acute Myeloid Leukemia by Inhibiting Glycolytic Function and Downregulating the Expression of DNA Replication Genes.

The BCL-2 inhibitor venetoclax has been widely used in the treatment of acute myeloid leukemia (AML); however, AML patients treated with venetoclax gradually develop resistance. The exportin-1 (XPO1) inhibitor selinexor can synergistically promote the antileukemia activity of venetoclax, but the mechanism remains unclear. Annexin V/7-aminoactinomycin D assays were used to examine the effects of a combination of venetoclax and selinexor (VEN+SEL) on AML cell lines and primary AML cells. RNA sequencing and oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) determinations by a Seahorse XF analyzer were employed to investigate the molecular mechanism of the toxicity of the VEN+SEL combination to AML cells. The cytotoxicity of NK cell combined with VEN+SEL combination was assessed in vitro using flow cytometry. VEN+SEL enhanced the apoptosis of AML cells (KG-1A and THP-1) and primary AML samples in vitro. The ECAR and OCR results demonstrated that the VEN+SEL combination significantly inhibited glycolytic function. RNA sequencing of THP-1 cells demonstrated that DNA replication-related genes were downregulated after treatment with the VEN+SEL combination. This study indicated that selinexor can synergistically enhance the antileukemia activity of venetoclax in AML cells in vitro by inhibiting glycolytic function and downregulating DNA replication-related genes. Based on our experimental data, combining selinexor with venetoclax is an appropriate advanced treatment option for AML patients.

AMPK activation induces immunogenic cell death in AML

AbstractSurvival of patients with acute myeloid leukemia (AML) can be improved by allogeneic hematopoietic stem cell transplantation (allo-HSCT) because of the antileukemic activity of T and natural killer cells from the donor. However, the use of allo-HSCT is limited by donor availability, recipient age, and potential severe side effects. Similarly, the efficacy of immunotherapies directing autologous T cells against tumor cells, including T-cell recruiting antibodies, chimeric antigen receptor T-cell therapy, and immune checkpoint inhibitors are limited in AML because of multiple mechanisms of leukemia immune escape. This has prompted a search for novel immunostimulatory approaches. Here, we show that activation of adenosine 5′-monophosphate–activated protein kinase (AMPK), a master regulator of cellular energy balance, by the small molecule GSK621 induces calreticulin (CALR) membrane exposure in murine and human AML cells. When CALR is exposed on the cell surface, it serves as a damage-associated molecular pattern that stimulates immune responses. We found that GSK621-treated murine leukemia cells promote the activation and maturation of bone marrow–derived dendritic cells. Moreover, vaccination with GSK621-treated leukemia cells had a protective effect in syngeneic immunocompetent recipients bearing transplanted AMLs. This effect was lost in recipients depleted of CD4/CD8 T cells. Together, these results demonstrate that AMPK activation by GSK621 elicits traits of immunogenic cell death and promotes a robust immune response against leukemia. Pharmacologic AMPK activation thus represents a new potential target for improving the activity of immunotherapy in AML.

IRAK-4 inhibition: emavusertib for the treatment of lymphoid and myeloid malignancies.

Several studies have identified mutations in the MYD88L265P gene as a key driver mutation in several B-cell lymphomas. B-cell lymphomas that harbor the MYD88L265P mutation form a complex with phosphorylated Bruton's tyrosine kinase (BTK) and are responsive to BTK inhibition. However, BTK inhibition in B-cell lymphomas rarely results in a complete response and most patients experience eventual disease relapse. Persistent survival signaling though downstream molecules such as interleukin 1 receptor-associated kinase 4 (IRAK-4), an integral part of the "myddosome" complex, has been shown to be constitutively active in B-cell lymphoma patients treated with BTK inhibitors. Emerging evidence is demonstrating the therapeutic benefit of IRAK-4 inhibition in B-cell lymphomas, along with possibly reversing BTK inhibitor resistance. While MYD88 gene mutations are not present in myeloid malignancies, downstream overexpression of the oncogenic long form of IRAK-4 has been found in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), particularly in AML and MDS that harbor mutations in splicing factors U2AF1 and SF3B1. These data suggest that the anti-leukemic activity of IRAK-4 inhibition can be exploited in relapsed/refractory (R/R) AML/MDS. In this review article, we discuss the currently available pre-clinical and clinical data of emavusertib, a selective, orally bioavailable IRAK-4 inhibitor in the treatment of R/R B-cell lymphomas and myeloid malignancies.

NKG2D-CAR memory T cells target pediatric T-cell acute lymphoblastic leukemia in vitro and in vivo but fail to eliminate leukemia initiating cells

IntroductionRefractory/relapsed pediatric acute leukemia are still clinically challenging and new therapeutic strategies are needed. Interactions between Natural Killer Group 2D (NKG2D) receptor, expressed in cytotoxic immune cells, and its ligands (NKG2DL), which are upregulated in leukemic blasts, are important for anti-leukemia immunosurveillance. Nevertheless, leukemia cells may develop immunoescape strategies as NKG2DL shedding and/or downregulation.MethodsIn this report, we analyzed the anti-leukemia activity of NKG2D chimeric antigen receptor (CAR) redirected memory (CD45RA-) T cells in vitro and in a murine model of T-cell acute lymphoblastic leukemia (T-ALL). We also explored in vitro how soluble NKG2DL (sNKG2DL) affected NKG2D-CAR T cells’ cytotoxicity and the impact of NKG2D-CAR T cells on Jurkat cells gene expression and in vivo functionality.ResultsIn vitro, we found NKG2D-CAR T cells targeted leukemia cells and showed resistance to the immunosuppressive effects exerted by sNKG2DL. In vivo, NKG2D-CAR T cells controlled T cell leukemia burden and increased survival of the treated mice but failed to cure the animals. After CAR T cell treatment, Jurkat cells upregulated genes related to proliferation, survival and stemness, and in vivo, they exhibited functional properties of leukemia initiating cells.DiscussionThe data here presented suggest, that, in combination with other therapeutic approaches, NKG2D-CAR T cells could be a novel treatment for pediatric T-ALL.

Simvastatin Preferentially Targets FLT3/ITD Acute Myeloid Leukemia by Inhibiting MEK/ERK and p38-MAPK Signaling Pathways.

The FLT3/ITD mutation exists in many acute myeloid leukemia (AML) patients and is related to the poor prognosis of patients. In this study, we attempted to evaluate the antitumor activity of simvastatin, a member of the statin class of drugs, in vitro and in vivo models of FLT3/ITD AML and to identify the potential mechanisms. Cell Counting Kit-8 (CCK-8) and Annexin V/propidium iodide (PI) staining kits were used to detect cell viability and apoptosis, respectively. Subsequently, Western blot and rescue experiment were applied to explore the potential molecular mechanism. In vivo anti-leukemia activity of simvastatin was evaluated in xenograft mouse models. In vitro experiments revealed that simvastatin inhibited AML progression in a dose- and time-dependent manner, while in vivo experiments showed that simvastatin significantly reduced tumor burden in FLT3/ITD xenograft mouse models. After simvastatin treatment of FLT3/ITD AML cells, intracellular Rap1 was downregulated and the phosphorylation levels of its downstream targets MEK, ERK and p38 were significantly inhibited. The rescue experiment showed that mevalonate, an intermediate product of the metabolic pathway of mevalonate, and its downstream geranylgeranyl pyrophosphate (GGPP) played a key role in this process. Finally, we demonstrate that simvastatin can induce apoptosis of primary AML cells, while having no effect on peripheral blood mononuclear cells from normal donors. Simvastatin can selectively and effectively eradicate FLT3/ITD AML cells in vitro and in vivo, and its mechanism may be related to the disruption of the HMG-CoA reductase pathway and the downregulation of the MEK/ERK and p38-MAPK signaling pathways.

Susceptibility of pediatric acute lymphoblastic leukemia to STAT3 inhibition depends on p53 induction.

Advances in the clinical management of pediatric B-cell acute lymphoblastic leukemia (B-ALL) have dramatically improved outcomes for this disease. However, relapsed and high-risk disease still contribute to significant numbers of treatment failures. Development of new, broad range therapies is urgently needed for these cases. We previously reported the susceptibility of ETV6-RUNX1+ pediatric B-ALL to inhibition of signal transducer and activator of transcription 3 (STAT3) activity. In the present study, we demonstrate that pharmacological or genetic inhibition of STAT3 results in p53 induction and that CRISPR-mediated TP53 knockout substantially reverses susceptibility to STAT3 inhibition. Furthermore, we demonstrate that sensitivity to STAT3 inhibition in patient-derived xenograft (PDX) B-ALL samples is not restricted to any particular disease subtype, but rather depends on TP53 status, the only resistant samples being TP53 mutant. Induction of p53 following STAT3 inhibition is not directly dependent on MDM2 but correlates with degradation of MDM4. As such, STAT3 inhibition exhibits synergistic in vitro and in vivo anti-leukemia activity when combined with MDM2 inhibition. Taken together with the relatively low frequency of TP53 mutations in this disease, these data support the future development of combined STAT3/ MDM2 inhibition in the therapy of refractory and relapsed pediatric B-ALL.

Targeting WEE1 kinase as a p53-independent therapeutic strategy in high-risk and relapsed acute lymphoblastic leukemia

BackgroundOutcomes for patients with relapsed acute lymphoblastic leukemia (ALL) are poor and there is a need for novel therapies to improve outcomes. Targeted inhibition of WEE1 with small-molecule inhibitor adavosertib (AZD1775) has emerged as a therapeutic strategy to sensitize cancer cells to DNA-damaging chemotherapeutics, particularly in the context of TP53-mutated tumors. However, WEE1 inhibition as a potential therapeutic strategy for patients with high-risk and relapsed ALL, including those with TP53 mutations, has not been definitively evaluated.MethodsAnti-leukemic effects of adavosertib were investigated using a relapsed TP53 isogenic cell model system, primary patient, and patient-derived ALL samples (n = 27) in an ex vivo co-culture model system with bone marrow-derived mesenchymal stem cells. Combination effects with drugs currently used for relapsed ALL were quantified by Excess over Bliss analyses. Investigations for alterations of cell cycle and apoptosis as well as related proteins were examined by flow cytometry and Western blot, respectively.ResultsOur study demonstrates the potent anti-leukemic activity of the clinically advanced WEE1 inhibitor adavosertib in a large majority (n = 18/27) of high-risk and relapsed ALL specimens at lower than clinically attainable concentrations, independent of TP53 mutation status. We show that treatment with adavosertib results in S-phase disruption even in the absence of DNA-damaging agents and that premature mitotic entry is not a prerequisite for its anti-leukemic effects. We further demonstrate that WEE1 inhibition additively and synergistically enhances the anti-leukemic effects of multiple conventional chemotherapeutics used in the relapsed ALL treatment setting. Particularly, we demonstrate the highly synergistic and cytotoxic combination of adavosertib with the nucleoside analog cytarabine and provide mechanistic insights into the combinational activity, showing preferential engagement of apoptotic cell death over cell cycle arrest. Our findings strongly support in vivo interrogation of adavosertib with cytarabine in xenograft models of relapsed and high-risk ALL.ConclusionsTogether, our data emphasize the functional importance of WEE1 in relapsed ALL cells and show WEE1 as a promising p53-independent therapeutic target for the improved treatment of high-risk and relapsed ALL.

A novel anticancer quinolone, (R)-WAC-224, has anti-leukemia activities against acute myeloid leukemia.

Approximately 60%-80% of patients who achieve complete remission eventually relapse after conventional chemotherapy and have poor prognoses despite the recent advances of novel anticancer agents. Continuing development of more effective novel treatments for acute myeloid leukemia (AML) is necessary. We developed (R)-WAC-224 (R-WAC), which is an anticancer quinolone, targeting topoisomerase II. This study evaluated the anti-leukemia potential of R-WAC or racemic WAC-224 (WAC) in vitro and in vivo. R-WAC significantly inhibited the human AML cell line proliferation (MV4-11, HL60, and KG1a), which was comparable to daunorubicin and cytarabine, not affected by P-glycoprotein overexpression. WAC did neither increase serum troponin-T nor decrease the crypt numbers in the small intestine, indicating WAC was less toxic than doxorubicin. R-WAC monotherapy demonstrated prolonged survival in the AML mice model and inhibited tumor growth in the MV4-11 xenograft mice model. Moreover, the combination of R-WAC and cytarabine demonstrated more active anti-leukemia effects than daunorubicin and cytarabine. Finally, R-WAC inhibited the colony-forming abilities using primary AML cells. These results indicate that R-WAC is a promising therapeutic agent for AML.