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Blood-based tumor liquid biopsies are promising as an alternative or complement to tissue biopsies due to their noninvasiveness, convenience, and safety, and there is still a great demand for the discovery of new biomarkers for these biopsies. Here, we present nanoscale distribution patterns of subcellular structures in platelets, as imaged by structured illumination superresolution fluorescence microscopy, as a new type of potential biomarker for tumor liquid biopsies. We established a standardized protocol for platelet sample preparation and developed an automated high-throughput image analysis workflow. We explored the diagnostic capability based on the statistical analysis of 200,000 superresolution images of individual platelets from a variety of tumor patients, benign mass patients, and healthy volunteers (n=163). Our results suggest that the nanoscale distribution patterns of α-granules in platelets have the potential to be biomarkers for several cancers, including glioma and cervical, endometrial and ovarian cancers, facilitating not only diagnosis but also therapeutic monitoring. This study provides a promising novel type of platelet parameter for tumor liquid biopsies at the subcellular level rather than the existing cellular or molecular level and opens up a new avenue for clinical applications of superresolution imaging techniques.

Background Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Its therapy has not significantly improved during the past four decades despite intense research efforts. New molecularly targeted therapies are in great need. The proto-oncogene c-Myc (MYC) is an attractive target due to its transactivation role in multiple signaling cascades. Deregulation of the MYC is considered one of a series of oncogenic events required for tumorigenesis. However, limited knowledge is available on which mechanism underlie MYC dysregulation and how long non-coding RNAs (lncRNAs) are involved in MYC dysregulation in AML. Methods AML microarray chips and public datasets were screened to identify novel lncRNA GAS6-AS1 was dysregulated in AML. Gain or loss of functional leukemia cell models were produced, and in vitro and in vivo experiments were applied to demonstrate its leukemogenic phenotypes. Interactive network analyses were performed to define intrinsic mechanism. Results We identified GAS6-AS1 was overexpressed in AML, and its aberrant function lead to more aggressive leukemia phenotypes and poorer survival outcomes. We revealed that GAS6-AS1 directly binds Y-box binding protein 1 (YBX1) to facilitate its interaction with MYC, leading to MYC transactivation and upregulation of IL1R1, RAB27B and other MYC target genes associated with leukemia progression. Further, lentiviral-based GAS6-AS1 silencing inhibited leukemia progression in vivo. Conclusions Our findings revealed a previously unappreciated role of GAS6-AS1 as an oncogenic lncRNA in AML progression and prognostic prediction. Importantly, we demonstrated that therapeutic targeting of the GAS6-AS1/YBX1/MYC axis inhibits AML cellular propagation and disease progression. Our insight in lncRNA associated MYC-driven leukemogenesis may contribute to develop new anti-leukemia treatment strategies.

Background The selective Janus-activated kinase inhibitor ruxolitinib (rux) is now widely used to treat myelofibrosis and polycythemia vera due to its remarkable effect of reducing splenomegaly and improving constitutional symptoms. With opportunistic infections secondary to rux constantly reported; however, an increasing number of studies have begun to investigate the mechanism and underlying immunosuppressive effect of rux. Case presentation We report two cases of tuberculosis (TB) in primary myelofibrosis patients during rux therapy. The first patient received rux soon after diagnosis, and tracheobronchial TB (TBTB) and bronchoesophageal fistula were found after 4 months. After discontinuation of rux, antituberculosis therapy (ATT) was introduced. The second patient initiated rux due to progressive splenomegaly after 7.5 years of interferon therapy and was diagnosed with disseminated TB after 2 months. He received ATT as well. His rux was maintained due to the high burden of systematic symptoms and splenomegaly. Both myelofibrosis and TB were well controlled in these patients. Conclusion This is the first case report that describes rux-related TBTB accompanied by a bronchoesophageal fistula. Through a review of the literature, we provide supporting evidence to the finding that intrinsic disorders of myeloproliferative neoplasms and rux-induced immunologic deregulation together lead to TB. We highlight the importance of screening for latent TB infection and timely chemoprophylaxis before rux therapy. Once TB is diagnosed during treatment, rux is recommended to be stopped and active ATT should begin quickly.

Chimeric antigen receptor (CAR) T cell therapy is a new frontier in cancer therapy. The toxicity of cytokine release syndrome (CRS) has become one of the major challenges that limits the wider use of CAR T cells to fight cancer. Exploration of CRS pathogenesis and treatment is becoming the main focus of ongoing studies. Myeloid-derived macrophages were found to play a critical role in CRS pathogenesis, and these cells mediate the major production of core cytokines, including IL-6, IL-1 and interferon (IFN)-γ. Colocalization of macrophages and CAR T cells was also identified as necessary for inducing CRS, and CD40L-CD40 signaling might be the key cell–cell interaction in the tumor microenvironment. Macrophages might also take part in endocrine and self-amplified catecholamine loops that can directly activate cytokine production and release by macrophages during CRS. In addition to tocilizumab and corticosteroids, several novel CRS therapies targeting macrophage-centered pathways have shown much potential, including GM-CSF blockade and administration of atrial natriuretic peptide (ANP) and α-methyltyrosine (metyrosine, MTR). In the present review, we summarized the role of macrophages in CRS and new developments in therapeutic strategies for CRS-associated toxicities.

Chimeric antigen receptor (CAR) T-cell immunotherapy is a new strategy for the treatment of refractory B-cell malignancies; therefore, the rapid and accurate quantification of CAR transgene copy number is essential. Real-time PCR was used for quantifying the copy number of chimeric antigen receptor transgene. Droplet digital PCR (ddPCR) is an absolute quantification method that does not require a standard curve. In this study, key performance parameters of the ddPCR and real-time PCR methods were assessed, including linearity, detection range, the lower limit of detection, repeatability, reproducibility, and accuracy, using a series of gradient diluted standards and clinical peripheral blood samples from CAR T-cell patients. The two platforms showed a good correlation for the standards (Pearson R2 = 0.9966; P

The aim of this study was to explore and compare routine blood features and pathological characteristics of bone marrow tissues in essential thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis, prefibrotic stage (prePMF) and overt fibrotic stage (overtPMF), and the correlation between common driver gene mutations and clinical manifestations of myeloproliferative neoplasms (MPN). Methods: We analyzed 259 MPN patients treated at Tongji Hospital of Huazhong University of Science and Technology from January 2016 to December 2020. Results: Among ET, PV, prePMF, and overtPMF, the median leukocyte counts of PV and prePMF were significantly higher than those of ET. The average hemoglobin level of overtPMF was significantly lower than that of ET, PV, and prePMF. ET and prePMF had higher platelet counts than PV and overtPMF, whereas ET had the lowest platelet distribution width. Regarding hematopoietic tissues in the bone marrow, enlarged megakaryocytes were easily found in ET, PV, and prePMF, whereas the average diameter of megakaryocytes in prePMF was smaller than in ET, and PV showed various sizes of megakaryocytes. An increased M/E ratio and dilation of sinus were seen more frequently in PMF. Additionally, JAK2-positive patients tended to have significantly higher leukocyte counts than CALR-positive patients in ET and PMF.

Myeloid-derived suppressor cells (MDSC) are a group of immature cells that produced by emergency myelopoiesis. Emerging evidences have identified the vital role of MDSC in cancer microenvironment, in which MDSC exerts both immunological and non-immunological activities to assist the progression of cancer. Advances in pre-clinical research have provided us the understanding of MDSC in cancer context from the perspective of molecular mechanism. In clinical scenario, MDSC and its subsets have been discovered to exist in peripheral blood and tumor site of patients from various types of cancers. In this review, we highlight the clinical value of MDSC in predicting prognosis of cancer patients and the responses of immunotherapies, therefore to propose the MDSC-inhibiting strategy in the scenario of cancer immunotherapies. Phenotypes and biological functions of MDSC in cancer microenvironment are comprehensively summarized to provide potential targets of MDSC-inhibiting strategy from the aspect of molecular mechanisms. Supplementary Information The online version contains supplementary material available at 10.1186/s40364-021-00333-5.

Secondary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome associated with infections, tumors and connective tissue disease. However rapid identification of the underlying infectious cause of HLH is challenging because traditional etiological diagnostics are time-consuming and sometimes fail to identify the pathogens. Metagenomic next-generation sequencing (mNGS) may be a potential optimal solution, which may help improve the clinical diagnosis of underlying infections in hematological diseases.A 28-year-old man presented with a 2-month history of intermittent fever and cytopenia. The HLH was diagnosed based on the manifestations of fever, splenomegaly, anemia, thrombocytopenia, hyperferritinemia, hyperglyceridemia, and elevated IL-2R levels. High-through-put sequencing analysis detected a GZMB mutation. While the initial detection of cultures and smears of tuberculosis was negative, TB infection was eventually identified by mNGS of blood sample. The symptoms rapidly abated during the initial administration of TB.The present case proposed that mNGS might be an effective diagnostic tool for diagnosing rare infectious cause of secondary HLH. GZMB mutation was first discovered to be present in secondary HLH.

High-dose chemotherapy followed by autologous stem cell transplantation (HDT-ASCT) is the standard of care for chemosensitive relapsed or refractory (R/R) aggressive B cell lymphoma. Patients with a positive positron emission tomography (PET) scan before ASCT have a poor prognosis, and those who fail to achieve a therapeutic response better than partial remission after salvage treatment are ineligible candidates for ASCT. We conducted this open-label single-arm prospective clinical study to evaluate the safety and efficacy of sequential infusion of CD19/22 chimeric antigen receptor (CAR) T cells following HDT-ASCT. Eligibility for this study included patients with R/R aggressive B cell non-Hodgkin lymphoma (B-NHL) with 18F-fluorodeoxyglucose-PET positivity and patients with stable or progressive disease after salvage chemotherapy. Between November 14, 2016, and August 15, 2019, 42 patients underwent HDT-ASCT followed by CD19/22 CAR T cell infusion. Grade 3 cytokine release syndrome (CRS) occurred in only 2 patients. Twenty-one percent of patients experienced any grade of neurotoxicity, 5% with severe grade 3. All cases of CRS and neurotoxicity were reversible. The overall response rate was 90.5% (95% confidence interval [CI], 77.4% to 97.3%). At a median follow-up of 24.3 months, the median progression-free survival (PFS) and overall survival were not reached. The 2-year PFS rate was 83.3 % (95% CI, 68.2% to 91.7%). No patients were found to be CD19- and CD22-negative at the time of progression; 97.1% and 68.6% of patients with ongoing complete remission (CR) had consistently detectable levels of CD19 and CD22 CAR transgene, respectively, at 3 months. The median time to onset of sustained B cell recovery was 8.2 months. The high durable CR rates and favorable safety profiles support the strong potential of the HDT-ASCT plus CD19/CD22 CAR T cell cocktail therapy for the suboptimal group of patients with R/R aggressive B-NHL who are less sensitive or fail salvage chemotherapy. These early data are encouraging and informative for future trials to further test the efficacy and safety of HDT-ASCT plus CAR T cell therapy in a larger population. © 2021 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Background Relapsed/refractory (R/R) multiple myeloma (MM) patients and primary plasma cell leukemia (PCL) have an unfavorable prognosis and no effective treatment. This study was designed to assess the safety and preliminary efficacy of a novel anti‐B‐cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cell in R/R MM and PCL. Methods Between February 22, 2017, and June 25, 2018, 28 R/R and two R/R primary PCL patients received a median dose of 11.2 × 106 CAR+ cells/kg. The subjects were refractory to a proteasome inhibitor and/or an immunomodulatory agent. Fludarabine and cyclophosphamide were given as lymphodepletion chemotherapy. Results Results for these 30 consecutive patients who received an anti‐BCMA CAR T cell infusion are reported. The patients had received a median of four prior lines of therapy. A total of 44 different types of adverse events were recorded, and hematologic toxic effects were the most common events of any grade during treatment. Hematologic toxic effects were also the most common events of grade 3 or higher. A total of 29 patients (96.7%) had cytokine release syndrome, which was of grade 1 or 2 in 24 patients (80%) and grade 3 in five patients (16.7%). Neurologic toxic effects only occurred in one patient (3.3%) and were of grade 1. The objective response rate was 90%, and the complete response rate was 43.3%. With a median follow‐up of 12.6 months, the median progression‐free survival (PFS) and overall survival were 5.2 months and 14.0 months. One of the two primary PCL achieved a complete response with a PFS of 307 days. The other patients achieved a very good partial response with a PFS of 117 days. Conclusions Anti‐BCMA CAR T cell treatment is safe and highly active in R/R multiple myeloma., We report the efficacy and safety of the infusion of anti‐BCMA CAR T cell treatment in with relapsed/refractory malignant plasma cell disease.Anti‐BCMA CAR T cell therapy exerted better safety and preliminary efficacy in relapsed/refractory multiple myeloma.Relapsed/refractory primary plasma cell leukemia may benefit from CAR T Cell treatment, although the duration of response is short.

B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T-cell therapies have shown efficacy in relapsed/refractory multiple myeloma (RRMM). Because the non-human originated antigen-targeting domain may limit clinical efficacy, we developed a fully human BCMA-specific CAR, CT103A, and report its safety and efficacy in a phase 1 trial. Eighteen consecutive patients with RRMM, including 4 with prior murine BCMA CAR exposures, were enrolled. CT103A was administered at 1, 3, and 6 × 106 CAR-positive T cells/kg in the dose-escalation phase, and 1 × 106 CAR-positive T cells/kg in the expansion cohort. The overall response rate was 100%, with 72.2% of the patients achieving complete response or stringent complete response. For the 4 murine BCMA CAR-exposed patients, 3 achieved stringent complete response, and 1 achieved a very good partial response. At 1 year, the progression-free survival rate was 58.3% for all cohorts and 79.1% for the patients without extramedullary myeloma. Hematologic toxicities were the most common adverse events; 70.6% of the patients experienced grade 1 or 2 cytokine release syndromes. No immune effector cell-associated neurotoxicity syndrome was observed. To the cutoff date, CAR transgenes were detectable in 77.8% of the patients. The median CAR transgene persistence was 307.5 days. Only 1 patient was positive for the anti-drug antibody. Altogether, CT103A is safe and highly active in patients with RRMM and can be developed as a promising therapy for RRMM. Patients who relapsed from prior murine BCMA CAR T-cell therapy may still benefit from CT103A. This trial was registered at http://www.chictr.org.cn as #ChiCTR1800018137.

B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T-cell therapies have shown efficacy in relapsed/refractory multiple myeloma (RRMM). Because the non-human originated antigen-targeting domain may limit clinical efficacy, we developed a fully human BCMA-specific CAR, CT103A, and report its safety and efficacy in a phase 1 trial. Eighteen consecutive patients with RRMM, including 4 with prior murine BCMA CAR exposures, were enrolled. CT103A was administered at 1, 3, and 6 × 106 CAR-positive T cells/kg in the dose-escalation phase, and 1 × 106 CAR-positive T cells/kg in the expansion cohort. The overall response rate was 100%, with 72.2% of the patients achieving complete response or stringent complete response. For the 4 murine BCMA CAR-exposed patients, 3 achieved stringent complete response, and 1 achieved a very good partial response. At 1 year, the progression-free survival rate was 58.3% for all cohorts and 79.1% for the patients without extramedullary myeloma. Hematologic toxicities were the most common adverse events; 70.6% of the patients experienced grade 1 or 2 cytokine release syndromes. No immune effector cell-associated neurotoxicity syndrome was observed. To the cutoff date, CAR transgenes were detectable in 77.8% of the patients. The median CAR transgene persistence was 307.5 days. Only 1 patient was positive for the anti-drug antibody. Altogether, CT103A is safe and highly active in patients with RRMM and can be developed as a promising therapy for RRMM. Patients who relapsed from prior murine BCMA CAR T-cell therapy may still benefit from CT103A. This trial was registered at http://www.chictr.org.cn as #ChiCTR1800018137.

Mitochondria are vital organelles that coordinate cellular energy homeostasis and have important roles in cell death. Therefore, the removal of damaged or excessive mitochondria is critical for maintaining proper cellular function. The PINK1-Parkin pathway removes acutely damaged mitochondria through a well-characterized mitophagy pathway, but basal mitochondrial turnover occurs via distinct and less well-understood mechanisms. Here we report that the MEKK3-MEK5-ERK5 kinase cascade is required for mitochondrial degradation in the absence of exogenous damage. We demonstrate that genetic or pharmacological inhibition of the MEKK3-MEK5-ERK5 pathway increases mitochondrial content by reducing lysosome-mediated degradation of mitochondria under basal conditions. We show that the MEKK3-MEK5-ERK5 pathway plays a selective role in basal mitochondrial degradation but is not required for non-selective bulk autophagy, damage-induced mitophagy, or restraint of mitochondrial biogenesis. This illuminates the MEKK3-MEK5-ERK5 pathway as a positive regulator of mitochondrial degradation that acts independently of exogenous mitochondrial stressors.

Background Chronic myelocytic leukemia (CML) may occasionally occur after organ transplantation or long-term chemotherapy for solid tumors; Solid tumors secondary to long-term chemotherapy and biotherapy in CML have also been reported. However, the concurrence of solid tumor with CML and extreme thrombocytosis in an untreated patient is seldom reported. Case presentation We describe a 61-year-old woman transferred to liver surgery department for the discovery of a large mass in the liver and an elevated plasmic AFP. She was initially diagnosed with liver cancer. Blood tests indicated a marked increase of platelets(2464x10 9 /L). The chromosome examination of bone marrow biopsy indicated the existence of t(9;22) translocation, fluorescence in situ hybridisation (FISH) and PCR were both positive for the Bcr-Abl rearrangement. The diagnosis of CML was made. She received hydroxyurea and imatinib to treat CML, and platelet-lowering therapy, and then underwent a liver biopsy, which suggestted a moderately-poorly differentiated adenocarcinoma, or might be a hepatic metastatic carcinoma. However, the patient refused further pathological examination and primary site screening for the tumor. She died six and a half months after discharge. Conclusion: Here, we describe a rare case of liver cancer concurrent with CML and extreme thrombocytosis in an old patient. However, the exact relationship between the two tumors is still unclear, and more cases are desired.

The rapid antitumor cytokine production and direct cytotoxicity confer invariant NKT (iNKT) cells ideal candidates for cancer therapy. However, the therapeutic potential of iNKT cells in T-cell malignant diseases remains elusive, as antigen presentation by T cells (T-T presentation) has been suggested to induce hyporesponsiveness of iNKT cells. In this study, we found discrepancies in iNKT cell responses against two T cell-origin cell lines (Jurkat and Molt-4). Human iNKT cells exhibited more intensive cytotoxicity and less efficient cytokine production in response to Fas-bearing Jurkat cells than those to the Fas-negative tumor cells (Molt-4 and myeloid-derived K562). The imbalanced cytokine/cytotoxicity responses of iNKT cells against Jurkat cells were CD1d-dependent and relied mostly on Fas/FasL interaction. The impairment in cytokine production could be overcome by Fas/FasL blocking antibodies and exogenous IL-2. Elevated CD1d levels as well as CD1d and Fas co-localization were found in T-cell lymphomas. However, defects in frequency and function of circulating iNKT cells were observed in the patients, which could be partly rescued by exogenous IL-2. Collectively, the Fas/FasL-dependent aberrant iNKT cell responses and the reversibility of the defects suggest the distinct iNKT cell manipulation in CD1d- and Fas-bearing T cell malignancies.

Background: Management of CML with TKIs is constrained by treatment resistance, which portends a poor prognosis particularly in pts failing 2 nd-generation TKIs. Cells with BCR-ABL1 T315I mutations are insensitive to 1 st- and 2 nd -generation TKIs, and compound BCR-ABL1 mutations complicate management with all TKIs (including 3 rd-generation ponatinib). Olverembatinib is a novel, potent, 3 rd-generation, orally active BCR-ABL1 TKI with promising activity against CML , largely irrespective of genotype and has a preliminary favorable safety profile. Methods: HQP1351-CC201 and HQP1351-CC202 are Chinese open, single-arm, multicenter phase 2 trials evaluating the safety and efficacy of olverembatinib in adults with TKI-resistant (BCR-ABL1 T351-mutated) CML-CP and CML-CP, respectively. Olverembatinib was administered at 40 mg orally on alternate days for 28-day cycles. The primary outcome measure is major cytogenetic response (MCyR) and major hematologic response (MaHR) by the end of Cycle 12 in CML-CP and CML-AP, respectively. Secondary study endpoints include : complete CyR (CCyR), complete hematologic response (CHR), major molecular response (MMR), progression-free survival (PFS), overall survival (OS), and safety, including treatment-related adverse events (TRAEs) and serious AEs (SAEs). Results: Baseline characteristics Study CC201 （CML-CP ） On the study cutoff date of August 25,2020, 41 pts were enrolled, of whom 32 (78%) completed ≥ 12 cycles and 21 (51.2%) were male. The median (range) follow-up was 13 (3.1-16.3) months, age was 47 (22-70) years, and interval from CML diagnosis to first olverembatinib dose was 5.31 (0.6-23.2) years. In all, 32 (78.1%) pts had received ≥ 2 prior TKIs and 9 pts withdrew because of progressive disease (PD), intolerance, or consent withdrawal before Cycle 12. Study CC202 （CML-AP ） On the cut-off date of July 27, 2020, 23 pts were enrolled, of whom 14 (61%) had completed ≥ 12 cycles and 18 (78.3%) were male. The median (range) follow-up was 13.5 (1.4-15.2) months, age was 41 (21-74) years, and interval from CML diagnosis to first olverembatinib dose was 4.96 (0.4-10.2) years. In all, 18 (78.3%) pts had received ≥ 2 prior TKIs, and 11 pts withdrew because of PD or intolerance before Cycle 12. Efficacy Study CC201 （CML-CP ） After ≥ 12 treatment cycles in pts without responses at baseline, all 31 (100%) experienced CHR (10 other pts had CHR at baseline); 31/41 (75.6%) MCyR; 28/41 (68.3%) CCyR; and 23/41 (56.1%) MMR (Figure 1). The median time to CHR was 1 (95% CI = 1.0-1.9) month, the median time to MCyR was 2.8 (95% CI = 2.8-5.6) months, and the median time to MMR was 6.5 (95% CI = 2.8 to not reached [NR]) months. At 12 months, the PFS rate was 89.3% (95% CI = 73.9%-95.8%), and the OS was 100% (95% CI = 100%-100%). Study CC202 （CML-AP ） After ≥ 12 treatment cycles in pts without responses at baseline, 17/23 (73.9%) experienced MaHR (65.2% CHR and 8.7% no evidence of leukemia [NEL]); 12/23 (52.2%) MCyR; 11/23 (47.8%) CCyR; and 9/23 (39.1%) MMR (Figure 1). The median time to MaHR was 2.8 (95% CI = 1.0-4.7) months, the median time to MCyR was 5.6 (95% CI = 2.00-NR) months, and the median time to MMR was 13.1 (95% CI = 5.6-NR) months. At 12 months, the PFS rate was 74.1% (95% CI = 48.2%-88.4%), and the OS was 91.3% (95% CI = 69.5%-97.8%). Safety Study CC201 （CML-CP ） Frequent TRAEs (all grades; grade 3-4; SAEs) included thrombocytopenia (70.7%; 48.8%; 7.3%), followed by anemia (61%; 26.8%; 2.4%), leukopenia (43.9%; 17.1%; 0), and neutropenia (36.6%; 19.5%; 0). Common nonhematologic TRAEs (all grades; G3-4) included skin pigmentation (56.1%, 0%) and elevations in creatine kinase (51.2%, 14.6%), ALT (39%, 2.4%) and AST (34.1%, 0) (Table 1). No deaths occurred. Study CC202 （CML-AP ） Common TRAEs (all grades; G3-4; SAEs) included thrombocytopenia (73.9%; 56.5%; 17.4%), anemia (60.9%; 34.8%; 13.0%), leukopenia (56.5%; 30.4%; 0), and neutropenia (26.1%; 21.7%; 0). Common nonhematologic AEs included skin pigmentation (69.6%), hypocalcemia (52.2%), proteinuria (47.8%), hypertriglyceridemia (56.5%), hyperphosphatemia (47.8%), hyperuricemia (21.7%), and arthralgia (34.8%), of which most were grade 1-2 (Table 2). Conclusions: Olverembatinib was efficacious and well tolerated when administered as monotherapy in pts with TKI-resistant CP-CML and AP-CML and the BCR-ABL1 T315Imutation. Internal study identifiers: HQP1351-CC201-CC202. ClinicalTrials.gov identifiers: NCT03883087 and NCT03883100. Figure 1 Figure 1. Disclosures Chen: Ascentage Pharma (Suzhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company. Niu: Ascentage Pharma (Suzhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company. Men: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company. Yang: Ascentage Pharma (Suzhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding.

INTRODUCTION HQP1351 (olverembatinib) is an orally active third-generation BCR-ABL TKI designed for treatment of the patients with CML, harboring T315I mutation, which confers resistance against all first- and second-generation TKIs. METHODS The TKI resistant CML patients harboring T315I mutations either in chronic-phase (CP), or in accelerated-phase (AP), were enrolled into two single-arm, multicenter, open-label pivotal studies: HQP1351-CC201 in and HQP1351-CC202, respectively. The HQP1351 was administered at 40 mg once every other day (QOD) for 28 consecutive days per cycle over 24 months. The primary objective was to evaluate efficacy by major cytogenetic response (MCyR) in patients with CML-CP and major hematologic response (MaHR) in CML-AP. Secondary objectives included safety, tolerability, and pharmacokinetics (PK). RESULTS Baseline characteristics Study CC201 (CML-CP) As of the study cut-off date of March 23, 2020, total 41 patients were enrolled, of whom 38 (92.7%) completed ≥ 6 cycles and 21 (51.2%) were male. Median (range) follow-up was 7.9 (3.1-11.1) months, median age was 47 (22-70) years old. Median (range) interval from CML diagnosis to first HQP1351 treatment was 5.31 (0.6-23.2) years, and 32 (78.1%) patients received ≥ 2 prior lines of TKI. Three patients withdrew from the study-due to progressive disease (PD), intolerance, or consent withdrawal. Study CC202 (CML-AP) As of the cut-off date of February 11, 2020, total 23 patients were enrolled, of whom 18 (78.3%) completed ≥ 6 cycles and 18 (78.3%) were male. Follow-up duration was 8.2 (1.4-9.6) months, median age was 41 (21-74) years old. Median interval from CML diagnosis to first dose of HQP1351 was 4.96 (0.4-10.2) years, and 18 (78.3%) patients received ≥ 2 prior TKIs. Five patients withdrew because of PD or intolerance before Cycle 6. Efficacy Study CC201 (CML-CP) Across a median follow-up of 7.9 months, the mean (95% CI) 3-month progression-free survival (PFS) was 100% (100-100%) and 6-month PFS 96.7% (78.6-99.5%) because of 1 PD. In 31 evaluable patients who did not have a complete hematologic response (CHR) at baseline, 30 (96.8%) achieved CHR. In 41 evaluable patients who did not have a complete CyR (CCyR) at baseline, 31 (75.6%) achieved MCyR, including 27 (65.9%) CCyR and 4 (9.8%) partial CyR (PCyR). Total 20 out of 41 (48.8%) evaluable patients achieved major molecular response (MMR; Figure 1). Study CC202 (CML-AP) Across a median follow-up of 8.2 months, the 3-month PFS was 100% (100-100%) and the 6- month PFS 95.5% (71.9-99.3%) because of 2 PDs. A total of 18 (78.3%) of 23 evaluable patients without MaHR at baseline achieved MaHR, including 14 (60.9%) with CHR. In the 23 evaluable patients without MCyR at baseline, 12 (52.2%) patients achieved MCyR, including 9 (39.1%) CCyR and 3 (13.1%) PCyR. A total of 6 out of 23 (26.1%) evaluable patients achieved MMR (Figure 1). HQP1351 was highly and durably efficacious in the CML patients with T315I mutation; the probability and depth of clinical response may increase with prolonged treatment period. Safety/tolerability Study CC201 Frequent treatment-related adverse events (TRAEs; all grades; G3-4) included thrombocytopenia (65.9%; 48.8%), followed by anemia (48.8%; 24.4%), leukopenia (46.3%; 12.2%), and neutropenia (36.6%; 19.5%). Common non-hematologic TRAEs of any grade included skin pigmentation (53.7%) as well as elevations in creatine kinase (48.8%), ALT (31.7%) and AST (26.8% which most were grade 1 - 2. No death occurred. Study CC202 Common TRAEs (all grades; G3-4; serious AE [SAE]) included thrombocytopenia (73.9%; 52.2%; 17.4%), anemia (65.2%; 39.1%; 13.0%), leukopenia (56.5%; 30.4%; 0%), and neutropenia (26.1%; 21.7%; 0%;). Common non-hematologic AEs included skin pigmentation (69.6%), hypocalcemia (52.2%), proteinuria (52.2%), hypertriglyceridemia (47.8%), hyperphosphatemia (43.5%), arthralgia (34.8%), and fatigue (26.1%), of which most were grade 1- 2. CONCLUSIONS HQP1351 has been shown highly efficacious in heavily TKI-pretreated patients with T315I-mutated CML-CP or CML-AP and was well tolerated. Registration: Clinicaltrials.gov identifier NCT03883087 (Study HQP1351-CC201) NCT03883100 (Study HQP1351-CC202). Disclosures Chen: Ascentage Pharma Group: Current Employment. Niu:Ascentage Pharma Group: Current Employment. Zeng:Ascentage Pharma Group: Current Employment. Men:Ascentage Pharma Group: Current Employment. Lu:Ascentage Pharma Group: Current Employment, Current equity holder in publicly-traded company. Wang:Ascentage Pharma Group: Current Employment. JI:Ascentage Pharma Group: Current Employment. Yue:Ascentage Pharma Group: Current Employment. Yang:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests. Zhai:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests.

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, yet constitutively active JAK2 mutants are able to drive selective expansion of particular lineage(s) in myeloproliferative neoplasm (MPN). The molecular basis of lineage specificity is unclear. Here, we show that three activating JAK2 mutants with similar kinase activities in vitro elicit distinctive MPN phenotypes in mice by differentially expanding erythroid vs granulocytic precursors. Molecularly, this reflects the differential binding of JAK2 mutants to cytokine receptors EpoR and GCSFR in the erythroid vs granulocytic lineage and the creation of unique receptor/JAK2 complexes that generate qualitatively distinct downstream signals. Our results demonstrate that activating JAK2 mutants can differentially couple to selective cytokine receptors and change the signaling repertoire, revealing the molecular basis for phenotypic differences elicited by JAK2 (V617F) or mutations in exon 12. On the basis of these findings, receptor-JAK2 interactions could represent new targets of lineage-specific therapeutic approaches against MPN, which may be applicable to other cancers with aberrant JAK-STAT signaling.

Background: Extramedullary myeloma (EMM) and primary plasma cell leukemia (pPCL) remains a treatment challenge even in the era of new drugs. The prognosis of EMM is inferior, and the median overall survival of patients who experience an extramedullary relapse is < 6 months. Chimeric antigen receptor (CAR)- B-cell maturation antigen (BCMA) has the potential for the treatment of EMM and PCL. Methods: Between February 22, 2017 and June 25, 2018, twenty-eight R/R MM patients (including fourteen EMM patients) and 2 pPCL patients received median dose of 11.2 × 106 CAR+ cells/kg (range, 5.4 – 25.0 × 106 CAR+ cells/kg). Patients had a median of 4 (range, 3 – 11) prior lines of therapy, including a proteasome inhibitor and/or an immunomodulatory agent. A lymphodepleting chemotherapy with cyclophosphamide 20mg/kg and fludarabine 25 mg/m2 daily on days -4 to -2 was administered before infusion of CAR-BCMA T cells on day 0. Findings: Hematologic toxic effects were the most common events of grade 3 or higher, including neutropenia (in 96.7% of the patients), lymphopenia (in 100%), anemia (in 76.7%), and thrombocytopenia (in 80.0%). Grade 3 or higher cytokine release syndrome and neurologic events occurred in 16.7% and 3.3% of the patients, respectively. The objective response rate (ORR) was 90%, and the complete response (CR) rate was 43.3%. One of 2 primary Plasma cell leukemia achieved CR, and another achieved a very good partial response (VGPR). With a median follow-up of 385 days, relapse occurred in nine of the 13 patients who had a CR. The median progression-free survival (PFS) and overall survivals (OS) were 158 days and 427 days. The median OS of patients with EMM/PCL was 243 days. Interpretation: CAR-BCMA T cell treatment is safe and effective in heavily treated R/R Multiple Myeloma with Extramedullary disease. Trial Registration: Chinese Clinical Trial Registry (http://www.chictr.org.cn. Number, ChiCTR OPC16009113). Funding Statement: This work is supported by the funding from the Key Program of the National Natural Science Foundation of China (81830008 and 81630006, to Dr. J.Z.), the Clinical research program of Huazhong University of Science and Technology affiliated Tongji Hospital (2019YBKY016, to Dr. J.Z.), the National Natural Science Foundation of China (81873452, to Dr. C.L.;81670152, to Dr. L.H.; 81600120, to Dr. N.W.). Declaration of Interests: The authors declare no competing financial interests. Ethics Approval Statement: This study was conducted in compliance with the Declaration of Helsinki. The study protocol was approved by the institutional review board of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.

Introduction HMPL-689 is a potent and highly selective small molecule inhibitor of phosphoinositide 3-kinase-delta (PI3Kδ). Despite available agents targeting the B-cell receptor (BCR) pathway, there remains a need for alternative therapies in the relapsed/refractory (R/R) setting due to agent-specific toxicities and suboptimal efficacy among lymphoma subtypes. This study (NCT03128164) is a phase 1, open-label, dose escalation and expansion study in China to assess the safety, pharmacokinetics (PK), and preliminary efficacy of HMPL-689 as a monotherapy in patients with R/R lymphomas. Here we present the preliminary results of the dose-escalation phase of the study. Methods In this dose escalation phase, patients with R/R lymphoma failed of standard therapy, at least 1 prior therapy, were eligible. The dose-escalation study consisted of cohort A (BID) and cohort B (QD), in which HMPL-689 was orally administered continuously on a 28-day cycle. The modified toxicity probability interval scheme-2 (mTPI-2) design was applied for the dose escalation and maximum tolerated dose (MTD) determination. Blood samples for PK and pharmacodynamics (PD) analyses were collected during Cycle 1 and Day 1 of each subsequent cycle. Results A total of 56 patients were enrolled, with 5 chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), 23 follicular lymphoma (FL), 7 marginal zone lymphoma (MZL), 9 mantle cell lymphoma (MCL), 9 diffuse large B cell lymphoma (DLBCL), and 3 Hodgkin's lymphoma (HL) patients (Table 1). The median age was 56 years (range 26-73). The median number of prior therapies was 3 (range 1-8), of which 39 patients had prior exposure to rituximab. 29 patients received HMPL-689 in cohort A (BID): 2.5 mg (n=3), 5 mg (n=9), 7.5 mg (n=8), and 10 mg (n=9), while 27 patients were in cohort B (QD): 5 mg (n=3), 10 mg (n=3), 20 mg (n=9), 30 mg (n=9), and 40 mg (n=3). Median duration of HMPL-689 therapy was 7.6 months (range 0.4 -Not reached [NR]). The most common Grade ≥3 non-hematologic treatment emergent adverse events (TEAEs) were pneumonia and hypertension. Grade ≥3 hematologic TEAEs were neutropenia (Table 2). No Grade 5 TEAE was reported. In cohort A, 4 dose limited toxicities (DLTs) were observed, including Grade 3 asymptomatic amylase (2 pts, 5 mg), Grade 4 hypercalcemia (1 pt, 10 mg), Grade 3 lipase increased (1 pt, 10 mg). In cohort B, 5 DLTs including Grade 3 skin maculopapular (1 pt, 20 mg), hypertriglyceridemia (1 pt, 30 mg), QT interval prolongation (1 pt, 30 mg) and rash (2 pts, 40 mg) were reported. Plasma PK data for the 5-30 mg QD and 2.5-10 mg BID multiple-dose regimens were determined. HMPL-689 drug exposures increased in a dose proportional fashion up to 30mg QD, as reflected in AUC and Cmax. The geometric mean AUCtau and Cmax at 30 mg QD in patients were approximately 2150 h•ng/mL and 260 ng/mL, respectively at steady state. The median Tmax was around 2 h and the arithmetic mean t1/2 was within the range of 5-10 hours, consistent across all dose levels. 51 out of the 56 patients had post-baseline tumor assessment, with 6 complete response (CR) (2 CLL/SLL, 4 FL), 21 partial responses (PR) (2 CLL/SLL, 5 MZL, 7 FL, 4 MCL, 3 DLBCL) and 18 stable disease (SD) (2 MZL, 9 FL, 4 MCL, 1 DLBCL, 2 HL). This resulted in 52.9% (27/51) objective response rate (ORR) in efficacy evaluable patients. The median time to response (TTR) and duration of response (DOR) were 3.5 months (1.8-8.4) and 6.4 months (0.7-NR), respectively (Table 3). One patient with FL who achieved CR (per post hoc independent radiologic review) was on treatment > 586 days. Final data quality control/verification is ongoing. As a result, 30 mg QD of HMPL-689 has been selected as recommended phase 2 dose (RP2D) based on overall safety and tolerability, PK/PD and preliminary efficacy data. Conclusions: HMPL-689 was well tolerated and the RP2D was determined to be 30 mg QD orally. It exhibited dose-proportional pharmacokinetics, a manageable toxicity profile, and promising single-agent clinical activity in R/R B-cell lymphoma patients. The dose expansion study is ongoing, evaluating the safety and efficacy of HMPL-689 in patients with R/R B-cell lymphoma. Acknowledgement 1. We would like to thank all patients and their families who participated in this trial; 2. We would like to thank all investigators, study coordinators and the entire project team. Disclosures Duan: Hutchison MediPharma Limitied: Current Employment. Yu:Hutchison MediPharma Limitied: Current Employment. Cai:Hutchison MediPharma Limitied: Current Employment. Su:Hutchison MediPharma Limited: Current Employment.

Background: Previous studies indicate that patients with relapsed/refractory multiple myeloma (RRMM) who receive BCMA-targeting CAR-T cells may achieve better remission but have a higher relapse rate. Persistence of CAR T cells post-infusion may be one determinant of the duration of response. Moreover, once the disease progresses again, the re-infusion of CAR-T cells is not effective. To solve this dilemma, we have developed a novel BCMA-targeting CAR-T (CT103A) with a lentiviral vector containing a CAR structure with a fully human scFv, CD8a hinger, and transmembrane, 4-1BB co-stimulatory and CD3z activation domains. Methods: ChiCTR1800018137 is a single-center and single-arm trial of CT103A in patients with RRMM (≥ 3 prior lines, including a proteasome inhibitor and an IMiD, or double refractory). The primary objectives are the incidence of adverse events (AEs), including dose-limiting toxicities (DLTs). The secondary objectives are the duration of clinical response, evaluation of minimal residual disease (MRD), progression-free and overall survival, and CAR-T cell persistence in blood. Between September 21, 2018, and August 1st, 2019, sixteen patients (including 4 patients having relapsed after being given a murine BCMA CAR-T and 5 patients having extramedullary disease and/or plasma cell leukemia) received CT103A in 3+3 dose-escalation trial (four doses at 1, 3, 6, 8 ×106/kg) after a conditioning chemotherapy regimen of cyclophosphamide and fludarabine. Median follow-up after CT103A infusion was 195 days (23 to 314 days) and all 16 patients were evaluable for initial (14 days) clinical response. Results: As of August 1st, 2019, the objective response rate was 100%, 6/16 patients achieved CR/sCR within two weeks post-infusion and all 8 patients surpassing 6 months achieved VGPR/CR/sCR. CR/sCR was 75%, and VGPR was 25% for these 8 patients, according to the IMWG Uniform Response Criteria for MM. In 4 patients who have participated in a prior CAR-T trial, three have achieved sCR, and 1 achieved VGPR. All 15 patients who could be evaluated for minimal residual disease (MRD) had MRD-negative status (≤10-4 nucleated cells by flow). The circulating CT103A cells were detected in the blood by flow and digital polymerase chain reaction, peaking at 14 days (ranging from 9 to 25), and remaining detectable in 12/16 patients, at the time of their last evaluation. Patient #1 (the first patient treated) has now exceeded 314 days of CART persistence, post-infusion. All sixteen patients developed cytokine release syndrome (according to ASBMT Consensus Grading for Cytokine Release Syndrome and Neurological Toxicity Associated with Immune Effector Cells: 10 Grade 1-2, 5 Grade 3,1 Grade 4). A grade 4 CRS appeared at the 6×106 /kg dose level and was considered as a dose-limiting toxicity DLT. No neurotoxicity was observed in all dose groups. One patient died of a lung infection 19 days post-infusion. Conclusions: Data from this early-stage clinical study showed the unparalleled safety and efficacy of CT103A in heavily pretreated R/R multiple myeloma patients. Highly active (ORR 100%) and rapid response within two weeks, suggests CT103A could be developed as a competitive therapy to treat patients with RRMM. Disclosures Hu: Nanjing Iaso Biotherapeutics Co. Ltd..: Employment. Yang:Nanjing Iaso Biotherapeutics Co.: Employment. Zhou:Nanjing Iaso Biotherapeutics Co. Ltd.: Other: Chairman of Advisory Committee of Science and Medicine .

// Zhiqiang Han 1,* , Xiaoli Wang 1,* , Liang Ma 2 , Lijuan Chen 3 , Min Xiao 1 , Liang Huang 1 , Yang Cao 1 , Jian Bai 1 , Ding Ma 1 , Jianfeng Zhou 1 and Zhenya Hong 1,4 1 Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China 2 Department of Urology, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China 3 Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China 4 Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China * These authors contributed equally to this work Correspondence: Zhenya Hong, email: // Keywords : STAT3; prostate cancer; tumor-initiating cell; ALDH; Stattic Received : May 07, 2014 Accepted : August 05, 2014 Published : August 06, 2014 Abstract Despite of tremendous research efforts to profile prostate cancer, the genetic alterations and biological processes that correlate with disease progression remain partially elusive. In this study we show that the STAT3 small molecule inhibitor Stattic caused S-phase accumulation at low-dose levels and led to massive apoptosis at a relatively high-dose level in prostate cancer cells. STAT3 knockdown led to the disruption of the microvascular niche which tumor-initiating cells (TICs) and non-tumor initiating cells (non-TICs)depend on. Primary human prostate cancer cells and prostate cancer cell line contained high aldehyde dehydrogenase activity (ALDH high ) subpopulations with stem cell-like characteristics, which expressed higher levels of the active phosphorylated form of STAT3 (pSTAT3) than that of non-ALDH high subpopulations. Stattic could singnificantly decreas the population of ALDH high prostate cancer cells even at low-dose levels. IL-6 can convert non-ALDH high cells to ALDH high cells in prostate cancer cell line as well as from cells derived from human prostate tumors, the conversion mediated by IL-6 was abrogated in the presence of STAT3 inhibitor or upon STAT3 knockdown. STAT3 knockdown significantly impaired the ability of prostate cancer cells to initiate development of prostate adenocarcinoma. Moreover, blockade of STAT3 signaling was significantly effective in eradicating the tumor-initiating and bulk tumor cancer cell populations in both prostate cancer cell-line xenograft model and patient-derived tumor xenograft (PDTX) models. This data suggests that targeting both tumor initiating and differentiated cell populations by STAT3 inhibition is predicted to have greater efficacy for prostate cancer treatment.

8013 Background: Previous studies indicate patients with relapsed/refractory multiple myeloma (RRMM) who receive high-dose BCMA-targeting CAR-T cells may achieve better remission but have worse adverse events. Moreover, once the disease progresses again, the re-infusion of CAR-T cells is not effective. To solve this dilemma, we have developed a novel BCMA-targeting CAR-T (CT103A) with a lentiviral vector containing a CAR structure with a fully human scFv, CD8a hinger and transmembrane, 4-1BB co-stimulatory and CD3z activation domains. Methods: ChiCTR1800018137 is a single-center and single-arm trial of CT103A in patients with RRMM. The primary objectives are to characterize the safety and tolerability in patients with R/R MM. The secondary objectives include evaluation of anti-myeloma activity, cytokines, CAR-T cell persistence, and pharmacokinetics. Between September 21, 2018, and January 21, 2019, nine patients (including 3 patients having relapsed after being given a murine BCMA CAR-T) received CT103A in 3+3 dose-escalation trial (three doses at 1, 3, 6 ×106/kg) after a conditioning chemotherapy regimen of cyclophosphamide and fludarabine. All Patients had received a median of 4 prior lines (range 3 - 5) of MM therapy. Results: At the time of the February 4, 2019 data analysis, the overall response rate was 100% (Table), and all patients had a rapid response within 14 days, with 67% (2/3) reaching CR/sCR at the lowest dose. The pharmacokinetics of CT103A were assessed by a digital polymerase chain reaction. Robust expansions were seen even at the lowest dosage level. In addition, Cmax and AUC0-28 reached levels comparable to reported CD19 CAR-T. In the first two dose groups, the grade of cytokine release syndrome (CRS) was 0 - 2. In the 6 ×106 /kg dose group, DLT had been observed in one patient. Conclusions: Data from this early-stage clinical study showed the unparalleled safety and efficacy of CT103A. Major AEs were transient, manageable, and reversible. three patients who relapsed the murine BCMA CAR-T were treated with CT103A, two patients achieved CR, and one patient achieved VGPR. 100% ORR and a rapid response within 2 weeks, suggests CT103A could be developed as a competitive therapeutic to treat patients with RRMM. Treatment Response (Case 1,5 and 7 are patients who relapsed the murine BCMA CAR-T). Clinical trial information: ChiCTR1800018137. [Table: see text]

CD19 CAR T-cell (CAR-19) therapy have elicited great clinical responses in B-cell malignancies. However, CD19-negative relapse has emerged as a major challenge for the long-term disease control post CAR-19 therapy and conferred a dismal outcome to these pts. Recently, co-targeting of CD19/CD20, CD19/CD22 or CD19/CD123 has been proposed as a strategic approach to overcome antigen loss relapse after CAR-19 therapy. However, the infusion scheme has not yet been defined, the efficacy to avoid antigen escape has not been tested, and its toxicity remains to be assessed. Furthermore, the impact of genetic abnormalities on the outcome post CAR T-cell therapy has not been fully elucidated. We conducted an open-label, single-center and single-arm pilot study of sequential infusion of third generation CAR T-cell "cocktail" comprising both anti-CD22 and anti-CD19 CAR T-cells, which was registered with Chinese Clinical Trial Registry (ChiCTR, number ChiCTR-OPN-16008526). Between March 2016 and January 2018, a total of 89 eligibility pts completed CAR-22/19 "cocktail" infusion and were included for analysis. Of 89 pts (median age 36 years; range, 9-71), 51 had refractory/relapsed (R/R) B-cell acute lymphoblastic leukemia (B-ALL) and 38 had R/R B-cell non-Hodgkin lymphoma (B-NHL). The cutoff date for data collection was April 30, 2018, with a median follow-up of 7.6 months (mos) for B-ALL and 5.4 mos for B-NHL. CAR-22 and CAR-19 cells were given separately in 2 divided doses (range, 1 to 4 for CAR-22, and 1 to 5 for CAR-19) with a dose-escalation infusion. Eighty-one pts firstly received CAR-22 infusion, and then CAR-19 infusion, while 8 pts received CAR-19 first and CAR-22 later. B-ALL pts received 2.73±1.19×106/kg CAR-22 and 2.61±1.45×106/kg CAR-19; B-NHL pts received instead higher doses, i.e. 5.28±2.44 ×106/kg CAR-22 and 5.14±2.06 ×106/kg CAR-19. Of the 50 evaluable B-ALL pts, 48 (96.0%) achieved complete remission (CR)/or CR with incomplete count recovery (CRi) at the day 30 assessment. By sensitive multi-parameter flow cytometry (MFC) assay, 47 (94.0%) achieved MRD-negative CR/CRi. At a median follow-up of 7.6 mos (range, 1.3 to 22.2), the progression-free survival (PFS) of all B-ALL pts was 12.0 mos and the overall survival (OS) was not reached. Of the 13 pts with Ph+ B-ALL, 6 of whom had T315I mutation, 11 (84.6%) achieved MRD-negative CR/CRi. The PFS was 15.4 months, and the OS was not reached at a median follow-up of 13.5 months (range, 3 to 22.5). Of the 6 pts with MLL translocations (mainly MLL-AF6), 66.7% were progression-free and the median PFS was not reached. However, among pts with Ph-like ALL, the PFS was only 4.6 months. A total of 23 pts relapsed, however, antigen loss of CD19 and CD22 was not detected by MFC. Forty-Seven pts (92.2%) experienced cytokine release syndrome (CRS) and 11 (21.6%) had severe CRS (≥ grade 3). Except for 1 pt, all the severe cases were reversible. Seven pts (13.7%) developed neurotoxicity. At a minimum follow-up of 3 months, the overall response rate (ORR) and CR rate in 36 evaluable B-NHL pts was 72.2% and 50%, respectively. At the cutoff date for data collection, 50.5% of all pts remained progression-free. A total of 8 pts (26.7%) had relapsed with a median PFS of 2.6 months. Re-biopsy and immunophenotyping was performed in 3 relapsed pts and antigen loss of CD19 or CD22 was not detected. At a median follow-up of 5.4 months (range, 3.0 to 16.2), both PFS and OS of all pts were not reached. Of the 9 pts with MYC translocation, 8 had a response and 7 maintained their responses with a median follow-up of 10.1 months, including all the 4 pts with "double-hit" lymphoma (concurrently carrying MYC and BCL2 or BCL6 translocations). Of the 10 pts with del(17p) or TP53 mutation, 8 (80.0%) had a clinical response. With a median follow-up of 5.3 months, the PFS was 3.3 months, and 45.0% of these pts were progression-free. All the B-NHL pts experienced CRS, but only 21.1% had severe (grade 3 or 4), but reversible CRS and 13.2% developed reversible neurotoxicity. In summary, our results indicated that infusion of third generation CD22/19 CAR T-cell "cocktail" is feasible and safe for pts with B-cell malignancies. Co-targeting to CD19/CD22 is a promising approach for overcoming antigen escape relapse of CAR-19 therapy, but this remain to be fully tested. The treatment response predictive value of distinct genetic subtypes for CAR T-cell therapy should be considered for selection of pts for personalized immunotherapy. Disclosures No relevant conflicts of interest to declare.

TMTP1, a 5-amino acid peptide NVVRQ, obtained by using the flagella peptide library screening in our previous studies, can be used for the labeling of malignant in situ and metastatic lesions, and even micro-metastases. In this study, TMTP1 was assessed for its ability to specifically target the malignant hematopoietic cells and metastatic lesions of hematological malignancies. FITC-TMTP1 was chemically synthesized. Immunofluorescence assay and competitive test were carried out to determine the specific binding capacity of TMTPl to hematological malignant cell lines, including HL60, k562, SHI-1, Jurkat, Raji, El-4 and umbilical cord blood mononuclear cells. Mononuclear cells were isolated from the bone marrow of healthy subjects and patients with chronic myeloid leukemia. Then the cells were co-cultured with TMTP1 or scrambled peptides and the binding and affinity of TMTP1 peptide to the primary cells of hematological malignancies were flow cytometrically analyzed. The binding specificity of TMTP1 to target hematological malignancies was measured in vivo by intravenous injection of FITC-conjugated TMTP1 into El-4 lymphoma-bearing mice. The results showed that TMTP1 specifically bound to the cells of a series of hematological malignancies, including HL60, k562, Jurkat, Raji, El-4 and chronic myeloid leukemia primary cells but not to bone marrow mononuclear cells from healthy subjects. By contrast, TMTP1 could bind to the metastatic foci of lymphoma originating from the EL-4 cell line while the scrambled peptide failed to do so. Moreover, the occult metastases could be identified, with high specificity, by detecting FITC-TMTP1. We are led to conclude that TMTP1, as a novel tumor-homing peptide, can serve as a marker for primary malignant and metastatic lesions for the early diagnosis of hematological malignances and a carrier of anticancer drugs for cancer treatment.

Tumor cells acquire the ability to proliferate uncontrollably, resist apoptosis, sustain angiogenesis and evade immune surveillance. Signal transducer and activator of transcription (STAT) 3 regulates all of these processes in a surprisingly large number of human cancers. Consequently, the STAT3 protein is emerging as an ideal target for cancer therapy. This paper reports the generation of an oncolytic adenovirus (M4), which selectively blocks STAT3 signaling in tumor cells as a novel therapeutic strategy. M4 selectively replicated in tumor cells and expressed high levels of antisense STAT3 complementary DNA during the late phase of the viral infection in a replication-dependent manner. The viral progeny yield of M4 in tumor cells was much higher than that of the parent adenoviral mutants, Ad5/dE1A. M4 effectively silenced STAT3 and its target genes in tumor cells while sparing normal cells and exhibited potent antitumoral efficacy in vitro and in vivo. Systemic administration of M4 significantly inhibited tumor growth in an orthotopic gastric carcinoma mouse model, eliminated abdominal cavity metastases and prolonged survival time. In summary, M4 has low toxicity and great potential as a therapeutic agent for different types of cancers.

Histone deacetylase was overexpressed in a variety of cancers and was closely correlated with oncogenic factors. The histone deacetylase inhibitor, trichostatin A (TSA) was shown to induce apoptosis in many cancer cells. However, the mechanism of TSA on induction of cancer cells apoptosis is poorly understood. This study was designed to characterize the global gene expression profiles before and after treatment of human leukemia cell line Molt-4 with TSA. Flow cytometry, MTT and DNA ladder were used to observe the effect of TSA on the apoptosis of MOLT-4 cells and normal human peripheral blood mononuclear cells (PBMC). Microarray, reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the difference of gene and protein expressions of Molt-4 cells after incubation of the cells with TSA. The results showed that TSA could induce Molt-4 apoptosis in dose- and time-dependent manners but spared PBMCs. Microarray analysis showed that after incubation with TSA for 9 h, 310 genes were upregulated and 313 genes were deregulated. These genes regulate the growth, differentiation and survival of cells. Among these genes, STAT5A was down-regulated by 80.4% and MYC was down-regulated by 77.3%. It was concluded that TSA has definite growth-inhibiting and apoptosis-inducing effects on Molt-4 cells in time- and dose-dependent manners, with weak cytotoxic effects on PBMCs at the same time. The mechanism of TSA selectively inducing apoptosis and inhibiting growth may be ascribed to the changes of pro-proliferation genes and anti-apoptosis genes.

Ovarian cancer is the leading cause of gynecologic cancer deaths among women. Although platinum-based chemotherapy is the first-line treatment for human ovarian cancer, chemoresistance remains a major obstacle to successful treatment, and there are currently no approved molecularly targeted therapies. Recent evidence indicates that signal transducer and activator of transcription-3 (STAT3) is a determinant of chemoresistance and is related to tumor recurrence in a large number of solid malignancies. In this study, we demonstrated that high levels of pSTAT3 were associated with chemoresistance in human ovarian cancer cells. Targeting STAT3 by siRNA technology markedly enhanced cisplatin-induced apoptosis in cisplatin-resistant ovarian cancer cells that expressed a high level of pSTAT3. Interleukin-6 (IL-6) could induce STAT3 activation in cisplatin-sensitive ovarian cancer cells and led to protection against cisplatin. The STAT3 siRNA treatment also blocked IL-6-induced STAT3 phosphorylation, resulting in the attenuation of the anti-apoptotic activity of IL-6. We found that the combination of cisplatin and STAT3 siRNA resulted in the collapse of the mitochondrial membrane potential, attenuated the expression of Bcl-xL and Bcl-2, and increased the release of cytochrome C and expression of Bax. Taken together, these results suggest that the pharmacological inhibition of STAT3 may be a promising therapeutic strategy for the management of chemoresistance in ovarian cancer.

Although dramatic clinical success has been achieved in acute promyelocytic leukemia (APL), the success of differentiating agents has not been reproduced in non-APL leukemia. A key barrier to the clinical success of arsenic is that it is not potent enough to achieve a clinical benefit at physiologically tolerable concentrations by targeting the leukemia cell differentiation pathway alone. We explored a novel combination approach to enhance the eradication of leukemia stem cells (LSCs) by arsenic in non-APL leukemia. In the present study, phosphatidylinositol 3-kinase /AKT/mammalian target of rapamycin (mTOR) phosphorylation was strengthened after As(2)S(2) exposure in leukemia cell lines and stem/progenitor cells, but not in cord blood mononuclear cells (CBMCs). propidium iodide-103, the dual PI3K/mTOR inhibitor, effectively inhibited the transient activation of the PI3K/AKT/mTOR pathway by As(2)S(2). The synergistic killing and differentiation induction effects on non-APL leukemia cells were examined both in vitro and in vivo. Eradication of non-APL LSCs was determined using the nonobese diabetic/severe combined immunodeficiency mouse model. We found that a combined As(2)S(2)/PI-103 treatment synergized strongly to kill non-APL leukemia cells and promote their differentiation in vitro. Furthermore, the combined As(2)S(2)/PI-103 treatment effectively reduced leukemia cell repopulation and eradicated non-APL LSCs partially via induction of differentiation while sparing normal hematopoietic stem cells. Taken together, these findings suggest that induction of the PI3K/AKT/mTOR pathway could provide a protective response to offset the antitumor efficacy of As(2)S(2). Targeting the PI3K/AKT/mTOR pathway in combination with As(2)S(2) could be exploited as a novel strategy to enhance the differentiation and killing of non-APL LSCs.

This study was designed to determine the impact of chrysoeriol on proliferation and cell cycle progression in the human multiple myeloma cell lines RPMI 8226 and KM3, and its related molecular mechanisms. Chryseoriol was identified by using the phosphorylated AKT-specific cytoblot high throughput assay. CCK-8 assay was employed to examine the growth inhibition rate and IC(50) (48 h) in peripheral blood mononuclear cells (PBMNCs), RPMI 8226 and KM3 cells treated with chrysoeriol at various concentrations. Cells were labeled with 5-6-carboxyfluorescein diacetate succinimidyl ester (CFSE), and the proliferation dynamics was detected by flow cytometry and analyzed with ModFit software. The cell cycles of RPMI 8226 and KM3 cells were measured by flow cytometry when the IC(50) concentration of chrysoeriol was adopted. The alterations in cell-cycle related proteins (Cyclin B1, Cyclin D1, p21) and proteins in PI3K-AKT-mTOR pathway were determined by Western blot analysis. The results showed the proliferation of multiple myeloma cells was significantly inhibited by chrysoeriol, resulting in cell cycle arrest in G(2)/M phase. Chrysoeriol could significantly reduce the expression of p-AKT (s473) and p-4eBP1 (t37/46) protein, meanwhile enhanced Cyclin B1 and p21 protein expression. Similar effects were not observed in PBMNCs from normal donors. It was concluded that chrysoeriol was a selective PI3K-AKT-mTOR pathway inhibitor. It restrained the proliferation of human multiple myeloma cells, but didn't affect proliferation of PBMNCs from normal donors. It might exhibit the cell cycle regulatory effect via the inhibition of PI3K-AKT-mTOR signal pathway.

Inhibitors of histone deacetylases (HDACIs) are a new generation of anticancer agents that selectively kill tumor cells. However, the molecular basis for their tumor selectivity is not well understood. we employed a genetic technique, named suppression of mortality by antisense rescue technique(SMART), to understand the molecular mechanism of apoptosis induced by HDACI and identify the genes that participate in this process. We identified several novel genes and demonstrated that HDACIs promote apoptosis through activation ubiquitin/proteasome by inducing ubiquitin B not ubiquitin C gene expression. According to degrading the mitochondria membrane protein Mcl-1 by activated ubiquitin/proteasome system, the mitochondria potential collapse and cell apoptosis. Further observation demonstrated that depletion of MCL-1 by RNA interference (RNAi) sensitizes Hela cells to TSA mediated mitochondria membrane potential collapse and apoptosis, and overexpression of MCL-1 confers apoptosis resistance.