Broad Range Of Hematologic Malignancies Research Articles

Hematopoietic Stem Cells Expressing Engineered CD45 Enable a Near Universal Targeted Therapy for Hematologic Diseases

Allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment for a broad range of hematological malignancies. Unfortunately, the current standard of care relies on untargeted toxic conditioning regimens associated with severe morbidity and mortality curtailing HCT's wider use. In the last decade, antigen-specific cell depleting therapies have revolutionized clinical practice in hematology. Although target selection remains complex, the pan-hematopoietic marker CD45, a protein tyrosine phosphatase which is exclusively expressed on all nucleated hematopoietic cells, could enable targeted depletion of the entire hematopoietic system including HSCs. Combined with HSCs engineered to be shielded from a CD45-targeting antibody-drug conjugate (ADC), this approach could create a near-universal strategy to replace a diseased hematopoietic system, irrespective of disease etiology or cell type. We performed alanine (Ala) scanning of the entire extracellular domain of CD45 to identify specific residues forming the epitopes for three different monoclonal antibodies (mAbs). Next, we screened base editors (BE) to generate amino acid substitutions that reduced or abolished the binding of specific mAbs, purified the first two extracellular regions of the most promising protein variants (CD45 D1-D2) and measured their binding to the mAbs by biolayer interferometry (BLI). Two antibodies showed drastically reduced or no binding to some of the CD45 variants. Some variants only reduced the binding of the mAb of the corresponding epitope while others affected the binding of mAbs from multiple epitopes located in more distant regions. These results suggested that several CD45 amino acid exchanges altered the receptor structure in a way that spanned at least two structural domains. This interpretation is supported by a substantial decrease in protein thermal stability, calling for caution when selecting suitable variants. Nevertheless, through a series of experiments and optimizations of the sgRNAs we identified a BE (ABE8e-SpRY + sgRNA49.3) that resulted in biophysically stable protein variants with editing of about 50% of hematopoietic stem and progenitor cells (HSPCs). These base edited HSPCs formed equal numbers of colonies as well as relative numbers of myeloid and erythroid colonies comparable to control HSPCs. Furthermore, they engrafted and supported multi-lineage cell differentiation in NBSGW host mice comparable to non-edited control HSPCs. Secondary transplantation experiments confirmed successful editing of long-term reconstituting HSCs (LT-HSCs). Collectively, these results indicate that the edited HSPCs were functionally intact. We further developed a humanized, Fc silent CD45-ADC designated CIM053-ADC. A single dose of 0.5mg/kg was very well tolerated by NBSGW mice engrafted with hCD34 + HSPCs and effectively depleted human hematopoietic cells including LT-HSCs. Next, we injected luciferase marked MOLM-14 cells, an aggressive myeloid tumor cell line, in mice priorly xenografted with edited or control human HSPCs. CIM053-ADC treatment was initiated 12 days after tumor injection when mice demonstrated clearly detectable, extensive and multilocular disease ( Fig. A). We deliberately assigned the mice with the most advanced disease to the ADC group. Mice then obtained saline or two doses of CIM053-ADC i.v.. Tumors were cleared in all CIM053-ADC treated mice whereas in all the mice that obtained saline the MOLM-14 cells rapidly grew and mice had to be euthanized 21-23 days post tumor injection ( Fig. B). Importantly, CIM053-ADC eliminated unedited hCD45 + non-tumor cells but base edited cells persisted in all tested organs. In summary, we developed a novel, highly potent tumor-selective therapy that spares a shielded hematopoietic system. Since CIM053-ADC targets the pan-hematopoietic marker CD45 this approach may not only be applicable to treat AML but most hematologic malignancies and possibly other diseases requiring HCT.

Top advances of the year: Leukemia.

In the year 2021, there were three new Food and Drug Administration approvals for all leukemia types: asciminib (Scemblix) for chronic myeloid leukemia, brexucabtagene autoleucel (Tecartus) for relapsed/refractory B-cell acute lymphocytic leukemia, and asparaginase erwinia chrysanthemi (recombinant)-rywn (Rylaze) for acute lymphocytic leukemia. This is down from 2017-2018 when eight new therapies were approved for acute myeloid leukemia alone. However, this decrease from prior years does not imply that little progress was made in our understanding or treatment of leukemias in 2021. Asciminib and brexucabtagene autoleucel, in particular, are representative of major developing trends. Asciminib, a targeted therapy, is only one of many drugs in development that are products of a bedside-to-bench approach fueled by new sequencing and other genetic technologies that have greatly increased our understanding of the biology behind hematologic diseases. Brexucabtagene autoleucel, an adoptive cell therapy, is the newest of several similar treatments for B cell-associated neoplasms, and it is representative of a massive push to develop novel immunotherapies for a broad range of hematologic malignancies. This commentary reviews the development of asciminib and brexucabtagene autoleucel and describes other major advances in the associated fields of targeted therapy and immunotherapy for leukemias.

Safety, Tolerability, Pharmacokinetics (PK) and Preliminary Antitumor Activity of the Cyclin-Dependent Kinase-9 (CDK9) Inhibitor AZD4573 in Relapsed/Refractory Hematological Malignancies: A Phase 1 First-in-Human Study

Introduction: CDK9, a serine/threonine kinase, regulates transcription elongation by phosphorylating RNA polymerase II at serine 2 (pSer2). Transient inhibition of CDK9 modulates expression of genes with short-lived transcripts and labile proteins, providing a potential therapeutic opportunity in tumors dependent upon oncogenes fitting these criteria. AZD4573 is a highly potent and selective CDK9 inhibitor that rapidly and preferentially decreases BCL2 family anti-apoptotic proteins MCL-1, BFL-1 as well as the oncogene MYC, inducing apoptosis in a broad range of human hematological malignant cell lines. AZD4573 monotherapy has shown antitumor effects associated with transient CDK9 inhibition in leukemia and lymphoma preclinical models. This multicenter, nonrandomized, open-label, phase 1 study (NCT03263637) was conducted to assess safety, tolerability, PK, pharmacodynamics (PD) and preliminary antitumor activity of AZD4573 in patients (pts) with relapsed or refractory hematological malignancies. Methods: The study comprised two parallel dose-escalation arms using a 3+3 design. Arm A included pts with diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, or multiple myeloma. Arm B included pts with acute myeloid/lymphocytic leukemia, chronic myelomonocytic/lymphocytic leukemia, or hairy cell leukemia. Pts must have received at least 2 prior lines of therapy for the current histology. In each arm, pts received AZD4573 IV on an 8-week cycle using an intra-patient ramp up starting at 6 mg and increasing in 3 mg increments to a target dose of 18 mg (cohort 1) or 12 mg (cohort 2). The initial dosing regimen was 2 days on/12 days off but was changed to once weekly (QW) in cohort 1. Pts in Cohort 3 received AZD4573 9 mg QW on a 4-week cycle including intra-patient ramp up (3 mg, 6 mg, 9 mg). The primary objectives were to assess safety and to determine the biologically effective dose/maximum tolerated dose (MTD). Adverse events (AE) were assessed using CTCAE v4.03. Secondary/exploratory endpoints included PK, PD, and antitumor efficacy. Results: Overall, 44 pts were treated and evaluated for safety and efficacy: 22 each in Arms A and B. Median age was 69.0 years (range, 26.0 - 84.0), 65.9% were male, and median number of prior lines of therapy was 3 (range, 1-11). At data cutoff (Sept 30, 2021), all pts had discontinued treatment, mainly due to disease progression (47.7%) or AEs (22.7%). All pts had treatment-emergent adverse events (TEAEs) which were serious in 88.6%. The most common TEAEs were diarrhea (59.1%), pyrexia (52.3%) and nausea (50.0%). The most common TEAE that led to discontinuation was pneumonia (4.5%). Treatment-related AEs (TRAEs) occurred in 93.2% of pts and were serious in 59.1%, but none led to death. The most common TRAEs were diarrhea (50%), nausea (47.7%) and tumor lysis syndrome (TLS, 40.9%: laboratory TLS, 38.6%; clinical TLS, 6.8%) (Table 1). Elevated liver enzymes were transient and resolved spontaneously. These increases were assumed to be mainly due to down-modulation of hepatic transporter proteins and reduced enzyme clearance rather than direct hepatocellular cytotoxicity (based on clinical, in-vitro and in-silico assessments). Regarding MTD, the 18 mg dose was not tolerated in either arm (clinical TLS and acute kidney injury in Arm A; hypotension and liver injury in Arm B). AZD4573 12 mg was tolerated in Arm A, but not tolerated in Arm B due to clinical TLS (n=2). The recommended Phase 2 dose (RP2D) was 12 mg QW in Arm A and 9 mg QW in Arm B. AZD4573 showed dose-proportional increases in AUC and Cmax, with moderate PK variability (~30-60% CV), and a t1/2 of ~5 hrs. Furthermore, AZD4573 led to dose-dependent reduction in pSer2 (>75%) and MCL-1 (>70%) in peripheral blood as evidence of target engagement and mechanism of action. Median duration of exposure was 10.1 wks in Arm A (range, 1.0-130.1) and 5.2 wks in Arm B (range, 1.0-35.7). In Arm A, there was 1 complete and 1 partial response in 17 DLBCL pts. Median overall survival was not reached in Arm A and was 8.8 months in Arm B, with 7 (31.8%) deaths in each. Conclusion: AZD4573 had manageable safety and a PK profile suitable for QW dosing in a broad range of hematologic malignancies including lymphoma and leukemia. The reduction in pSer2 and MCL-1 supports the RP2D of 12 mg for lymphoma and 9 mg for leukemia. A combination study of AZD4573 and acalabrutinib (NCT04630756) is ongoing to further examine the DLBCL response signal seen in this study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immunomodulatory Effects of Bendamustine in Hematopoietic Cell Transplantation.

Simple SummaryBendamustine is a chemotherapeutic agent used to treat a variety of cancers. It has recently been used in the context of allogeneic hematopoietic cell transplantation (HCT), a treatment mostly used to treat blood cancers. Given before or after transplantation of donor blood or bone marrow cells, bendamustine has been shown to reduce the side effects of the transplant, including graft-versus-host disease, where the donated cells attack the recipient’s tissues, while also promoting the anti-cancer effects of the transplant. These are exciting findings and show that bendamustine may be used to influence the immune system, called immunomodulation, in a beneficial manner. We report our research and review the available literature outlining these immunomodulatory effects of bendamustine, in hopes that it will promote further investigations utilizing this agent in allogeneic transplants, ultimately improving patient outcomes.Bendamustine (BEN) is a unique alkylating agent with efficacy against a broad range of hematological malignancies, although investigations have only recently started to delve into its immunomodulatory effects. These immunomodulatory properties of BEN in the context of hematopoietic cell transplantation (HCT) are reviewed here. Pre- and post-transplant use of BEN in multiple murine models have consistently resulted in reduced GvHD and enhanced GvL, with significant changes to key immunological cell populations, including T-cells, myeloid derived suppressor cells (MDSCs), and dendritic cells (DCs). Further, in vitro studies find that BEN enhances the suppressive function of MDSCs, skews DCs toward cDC1s, enhances Flt3 expression on DCs, increases B-cell production of IL-10, inhibits STAT3 activation, and suppresses proliferation of T- and B-cells. Overall, BEN has a broad range of immunomodulatory effects that, as they are further elucidated, may be exploited to improve clinical outcomes. As such, clinical trials are currently underway investigating new potential applications of BEN in the setting of allogeneic HCT.

Simultaneous Kinase Inhibition with Ibrutinib and BCL2 Inhibition with Venetoclax As a Therapeutic Strategy for Acute Lymphoblastic Leukemia

Simultaneous Kinase Inhibition with Ibrutinib and BCL2 Inhibition with Venetoclax As a Therapeutic Strategy for Acute Lymphoblastic Leukemia

New Treatments for Relapsed Hematologic Malignancies After alloHCT

New Treatments for Relapsed Hematologic Malignancies After alloHCT

SL-401, a Targeted Therapy Directed to the Interleukin-3 Receptor (CD123), and SL-801, a Reversible Inhibitor of Exportin-1 (XPO1), Display Synergistic Anti-Tumor Activity Against Hematologic Malignancies in Vitro

Background: Novel combination therapies have shown success in combating tumor heterogeneity and drug resistance. SL-401 is a targeted therapy directed to the interleukin-3 receptor (CD123), which is overexpressed on numerous hematologic malignancies. SL-401 has demonstrated high single agent response rates in an ongoing Phase 2 trial of blastic plasmacytoid dendritic cell neoplasm (BPDCN) and is also being evaluated in the clinic for additional cancers, including acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs) as a single agent, and multiple myeloma (MM) in combination with other agents. While SL-401 has demonstrated robust single agent clinical activity in patients with BPDCN, its unique mechanism of action and non-overlapping side effect profile with other agents may lend itself to combination therapy as well. Another class of drugs that has demonstrated clinical activity against several hematologic and solid malignancies is Exportin-1 (XPO1) inhibitors. SL-801 is a novel oral small molecule that reversibly inhibits XPO1 and has shown potent in vitro and in vivo anti-tumor activity against a broad range of hematologic and solid malignancies. SL-801 is currently being evaluated in a Phase 1 trial of patients with advanced solid tumors, and a Phase 1 trial in advanced hematologic cancers is planned. Here, we investigated the in vitro effect of combination treatment of SL-401 and SL-801 against cell lines of chronic myeloid leukemia (CML), AML, MM, and Hodgkin's lymphoma (HL).Methods: The human K562 CML cell line, MV4-11 AML cell line, RPMI-8226 MM cell line, and L-428 HL cell line were treated with varying concentrations of SL-401 and SL-801 alone or in combination for 48 hours. Cell viability was assessed by the CellTiter Glo in vitro cytotoxicity assay. Combination index (CI) values were calculated using CompuSyn software by the method of Chou and Talalay, and treatment was considered to be synergistic when CI < 1. Caspase activation was measured using the Caspase-Glo 3/7 assay, and lactate dehydrogenase (LDH) release was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay.Results: As single agents, SL-401 and SL-801 demonstrated anti-tumor activity in all four cell lines tested. MV4-11 cells were the most sensitive to both drugs, with an IC50 of 34 pM for SL-401 and 21 nM for SL-801. In the other cell lines, the IC50s for SL-401 were 17 nM in K562 cells, 25 nM in RPMI-8226 cells, and 100 nM in L-428 cells, and the IC50s for SL-801 were 99 nM in K562 cells, 51 nM in RPMI-8226 cells, and 494 nM in L-428 cells. When combined with each other, SL-401 and SL-801 potently inhibited cell growth in all cell lines, and CI calculations indicated that the interaction between the two drugs was synergistic at most dose combinations. Notably, CI values < 0.3 were observed in MV4-11 and L-428 cells, indicative of strong synergy. Consistent with these observations, the combination of SL-401 and SL-801 also induced higher levels of caspase activation and LDH release in MV4-11 and L-428 cells than either drug alone.Conclusion: These findings demonstrate that SL-401 and SL-801, when combined, act synergistically in their in vitro anti-tumor activity against CML, AML, MM, and HL cells. Investigations into the molecular mechanisms underlying the observed synergy are in progress. These promising results provide rationale for further development of SL-401 and SL-801 combination therapy in the treatment of a broad range of hematologic malignancies. DisclosuresGionco:Stemline Therapeutics, Inc.: Employment. Chen:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Lindsay:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Macri:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

Sex chromosome loss and the pseudoautosomal region genes in hematological malignancies.

Cytogenetic aberrations, such as chromosomal translocations, aneuploidy, and amplifications, are frequently detected in hematological malignancies. For many of the common autosomal aberrations, the mechanisms underlying their roles in cancer development have been well-characterized. On the contrary, although loss of a sex chromosome is observed in a broad range of hematological malignancies, how it cooperates in disease development is less understood. Nevertheless, it has been postulated that tumor suppressor genes reside on the sex chromosomes. Although the X and Y sex chromosomes are highly divergent, the pseudoautosomal regions are homologous between both chromosomes. Here, we review what is currently known about the pseudoautosomal region genes in the hematological system. Additionally, we discuss implications for haploinsufficiency of critical pseudoautosomal region sex chromosome genes, driven by sex chromosome loss, in promoting hematological malignancies. Because mechanistic studies on disease development rely heavily on murine models, we also discuss the challenges and caveats of existing models, and propose alternatives for examining the involvement of pseudoautosomal region genes and loss of a sex chromosome in vivo. With the widespread detection of loss of a sex chromosome in different hematological malignances, the elucidation of the role of pseudoautosomal region genes in the development and progression of these diseases would be invaluable to the field.

Abstract 4711: Broad anti-tumor activity of a novel BET bromodomain inhibitor

Abstract Inhibitors against the bromodomain and extra terminal domain (BET) family of proteins have been pursued as promising oncology agents based on growing understanding of epigenetic control of disease processes. Through scaffold-based and crystallography-guided drug design, we discovered PLX51107, a potent and selective small molecule inhibitor of the BET family bromodomains. PLX51107 is structurally unrelated to the benzodiazepines such as JQ1, I-BET762, and OTX015 and other published BET inhibitors. PLX51107 exhibits low nanomolar potency in blocking interactions mediated by the four BET family proteins BRD2, BRD3, BRD4, and BRDT. Pharmacologic inhibition of BET proteins by PLX51107 suppresses the transcription of genes essential for tumor growth and survival and leads to selective killing of cancer cell lines across a broad range of hematologic malignancies (e.g. leukemia, lymphoma and multiple myeloma). A subset of solid tumors (e.g. melanoma and SCLC) is also sensitive to growth inhibition by the BET inhibitor PLX51107. Novel biomarkers in these diseases have been identified. PLX51107 is well tolerated and has sufficient potency and oral bioavailability to demonstrate in vivo efficacy in animal models of a variety of tumor types, representing both hematologic and solid tumors of diverse genetic backgrounds. In combination studies, PLX51107 showed potential to improve the efficacy (response rates and duration of response) of other anticancer treatments without increased toxicity. These results support further development of PLX51107 as an epigenetic-based therapy for a variety of cancer indications. Citation Format: Yan Ma, Ben Powell, Jiazhong Zhang, Ying Zhang, Heidi Carias, Ullrich Schwertschlag, Gaston Habets, Prabha Ibrahim, Wayne Spevak, Chao Zhang, Gideon Bollag. Broad anti-tumor activity of a novel BET bromodomain inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4711.

Abstract 4516: Evaluating the chromatin state as a predictive biomarker for BET inhibitor sensitivity in hematological malignancies

Abstract The BET (bromodomain extra-terminal) family of proteins are a group of epigenetic regulators that control gene transcription by reading acetylated lysines on histone tails and recruiting transcription complexes. The various BET inhibitors developed show strong anti-tumor effects against a broad range of hematologic malignancies. The BET inhibitor JQ1 shows strong anti-proliferative activity in sub-sets of multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and lymphoma cell line models. At present, however, predictive biomarkers of BET inhibitor sensitivity have not been identified. We hypothesize that the chromatin state characterized by various histone post-translational modifications (acetylation, methylation, etc.), can drive sensitivity to BET inhibition. By examining basal expression of epigenetic marks in panels of sensitive and resistant heme cell line models and further validating these histone modification trends in primary AML and multiple myeloma cultures, we may be able to identify predictive biomarkers of BET inhibitor sensitivity in hematological malignancies. Citation Format: Joseph Castillo, Ryan Raisner, Karen Gascoigne, Gary Dos Santos, Emer Clarke, Alex Huang, Mark Lackner, Zineb Mounir. Evaluating the chromatin state as a predictive biomarker for BET inhibitor sensitivity in hematological malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4516.

Graft-versus-host disease versus graft-versus-leukemia.

Graft-versus-host disease (GVHD) is a significant clinical problem after allogenic hematopoietic cell transplantation (HCT) associated with substantial morbidity and mortality that limits the potential utility of transplantation. Associated with GVHD is the well-recognized phenomenon of the graft-versus-leukemia (GVL) effect that results in reduced risk of disease relapse. GVL effects have been observed after treatment for a broad range of hematological malignancies. Both GVHD and GVL are the results of T cell-effector functions that frames a major question in the field of how linked are these two phenomena. A major goal of basic science and translational research has been to develop strategies to reduce the risk of GVHD while maintaining or enhancing GVL. In this review, a number of different strategies developed from preclinical animal models will be explored with a focus on those approaches that have been extended to the clinic in an attempt to achieve this goal. Needless to say, there is no proven strategy; however, with the use of modern technology and clinical translation, there has been substantial progress toward this goal of reducing the risks of GVHD while promoting and enhancing GVL responses.

Results of a Two-Arm Phase II Clinical Trial Using Post-Transplantation Cyclophosphamide for Prevention of Graft-Versus-Host Disease in Haploidentical and Mismatched Unrelated Donors Hematopoietic Stem-Cell Transplantation

▪Allogeneic stem cell transplantation offers curative therapy for many patients (pts) with high-risk hematologic malignancies. Donor availability remains a major limitation for many pts. The introduction of high-dose post-transplant cyclophosphamide (PTCy) has significantly improved the outcomes of pts undergoing haploidentical (HAPLO) stem cell transplants. The choice between a HAPLO or a one-antigen HLA mismatched unrelated donor (9/10 MUD) for pts lacking an HLA-matched donor remains unclear.Methods:We conducted a prospective non-randomized phase 2 clinical trial with two parallel arms, HAPLO (n=60) and 9/10 MUD (n=46) transplants, for pts with advanced hematologic malignancies or aplastic anemia who lacked an HLA-matched unrelated donor type at 10 loci (HLA-A, -B, -C, -DRB1, and -DQB1) using a MEL-based reduced-intensity conditioning regimen. The regimen included a single intravenous dose of MEL 140 mg/m2 (day -7), thiotepa 5 mg/kg (day -6), and four daily IV doses of fludarabine 40 mg/m2 (day -5 to day -2) (FM140). Thiotepa was intermittently available and was replaced by total body irradiation at a dose of 2 Gy on day -1. Pts >55 years (yr) old or with significant comorbidities received a lower MEL dose (100 mg/m2) (FM100). All pts with CD20-positive lymphoma received rituximab (375 mg/m2) on days -13, -6, +1 and +8. GVHD prophylaxis consisted of PTCy 50 mg/kg on day +3 and +4, and tacrolimus and mycophenolate for 6 and 3 months (mo), respectively. The stem cell source was unmodified bone marrow for both arms.Results:Patient characteristics are shown in Table 1. The median follow-up duration was 24 mo in the HAPLO arm and 29 mo in the 9/10 MUD arm. The cumulative incidence (CI) of neutrophil (ANC) recovery at day 45 was 95% and 98% in the HAPLO and 9/10 MUD arm, respectively. The median time to ANC recovery was 18 days in both arms; the median time to platelet recovery was 25 days in the HAPLO arm and 28 days in the 9/10 MUD arm. Primary graft failure developed in two pts in the HAPLO arm (one due to anti-donor HLA antibodies) and one patient in the 9/10 MUD arm. One pt in both arms developed mixed donor chimerism at day 100; otherwise, all pts in both arms achieved full (>95%) donor chimerism. Bone marrow was the graft source in all pts except 2 in the HAPLO arm and 8 in the 9/10 MUD arm who received a peripheral blood graft. The 1-yr overall and progression free survival were 70% and 60%, respectively, in the HAPLO arm (Fig. 1A) and 60% and 47%, respectively, in the 9/10 MUD arm (Fig. 1B). Day 100 CI of grade II-IV aGVHD and III-IV aGVHD were 28% and 3%, respectively, in the HAPLO arm versus 33% and 13%, respectively, in the 9/10 MUD arm; the 2-yr CI of chronic extensive GVHD was 13% and 14% in the two groups, respectively. The 1-yr CI of non-relapse mortality was 21% in the HAPLO arm and 31% in the 9/10 MUD arm, while the 1-yr relapse rate was 19% and 25% in the two groups, respectively.Conclusions:This study establishes PTCy, tacrolimus, and mycophenolate as an effective regimen for GVHD prevention in mismatched transplantation using both haploidentical and mismatched unrelated donor sources. Melphalan-based reduced-intensity conditioning is an effective regimen for a broad range of hematologic malignancies. Prospective randomized studies comparing haploidentical and unrelated donor sources are needed.Table 1HAPLO (n=60)9/10 MUD (n=46)Median Age, years (Range)45 (20-63)51 (20-64)Sex (M/F)29/3123/23KPS³9053 (88%)40 (87%)<907 (12%)6 (13%)HCT-CI0-350 (83%)38 (83%)>310 (17%)8 (17%)Disease Risk Index*Very high5 (8%)3 (7%)High18 (30%)15 (33%)Intermediate29 (48%)12 (26%)Low8 (13%)12 (26%)NA04 (9%)**Conditioning RegimenFM10020 (33)18 (39%)FM14040 (67%)28 (61%)DiagnosisAML/MDS33 (55%)18 (39%)ALL7 (11%)5 (11%)Lymphoma10 (17%)13 (28%)Others10 (17%)10 (22%)Disease StageAcute LeukemiaCR1/CR224 (66%)9 (56%)CR3 or higher/ CRpx6 (17%)5 (31%)Active disease6 (17%)2 (13%)LymphomaCR3 (30%)8 (62%)PR5 (50%)3 (23%)Chemoresistant2 (20%)2 (15%)*Disease Risk Index by Armand et al; xCRp: Complete Remission with incomplete count recovery; **Patients had aplastic anemia. [Display omitted] DisclosuresBrammer:Celgene: Research Funding. Lee:Ziopharm: Equity Ownership; Cyto-Sen: Equity Ownership; Intrexon: Equity Ownership. Rezvani:Pharmacyclics: Research Funding. Alousi:Therakos, Inc: Research Funding.

The Beta-Subunit Of Heterotrimeric G Proteins Harbors Gain-Of-Function Mutations In Multiple Hematologic Malignancies

To identify new oncogene alleles directly from primary tumor specimens, we generate and screen cDNA libraries from patient samples for gain-of-function alterations that can substitute for cytokine signaling in cytokine-dependent cells. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive leukemia of plasmacytoid dendritic cells with a dismal prognosis. No driver oncogenes have been identified in cases of BPDCN. Screening of a cDNA library generated from a BPDCN resulted in multiple cytokine-independent clones that expressed a full-length transcript of GNB1 with a K89E mutation. GNB1 encodes a beta subunit of the heterotrimeric G-protein, a binding complex that transduces signals from G-protein coupled receptors to multiple downstream pathways. Gain-of-function mutations have been reported in alpha subunits of the G-protein, including GNAQ/GNA11 in uveal melanoma and GNAS in pituitary tumors, however, the contributions of beta subunits to cancer remains undefined. To investigate downstream signaling from GNB1 K89E, we performed gene expression profiling and mass spectrometry (MS)-based phosphoproteomics and found significant activation of RAS/MAPK and PI3K/AKT pathways in GNB1 K89E-expressing cells compared to isogenic cells expressing wild-type GNB1. ERK and AKT activation by GNB1 K89E were confirmed by western blotting. To target GNB1 K89E signaling, we screened kinase inhibitors using a multiplex assay of small molecules and found selective sensitivity of GNB1 K89E cells to MEK and pan-PI3-kinase inhibitors. To assay the transforming effects of GNB1 K89E in vivo, we transduced GNB1 (wild-type or K89E) into bone marrow from Cdkn2a-deficient donors after 5-FU treatment and transplanted into wild-type recipients. We opted to utilize Cdkn2a-deficient donors as the loss of CDKN2A is common in cases of BPDCN. Within 4 months after transplantation, all mice (n=10) that received bone marrow transduced with GNB1 K89E developed a lethal malignancy characterized by pancytopenia and massive hepatosplenomegaly. Spleens were infiltrated by large, spindly cells with extensive dendritic projections, as well as extensive fibrosis that completely effaced the normal splenic architecture. The cells were negative for T-cell (CD2, CD3) and B-cell (CD19, B220) markers but positive for the dendritic cell/macrophage markers MAC-2 and MAC-3. Further characterization by flow cytometry demonstrated that the cells infiltrating the spleen were CD8, CD103, MHC class II, CD26, FLT3 and CD11c positive, consistent with neoplastic dendritic cells. Serial transplantation of splenic cells from five different GNB1 K89E-transplanted mice into secondary wild-type recipients resulted in 100% fatality within 50 days. We searched published datasets from exome, transcriptome and whole genome sequencing of hematologic malignancies for GNB1 mutations. We identified one case of K89E in B-cell acute lymphoblastic leukemia (ALL), four cases with I80T/N in chronic lymphocytic leukemia or B-cell lymphomas, six cases with K57E/T in myeloid neoplasms, and D76G in T-cell ALL. Expression of any of these alleles but not wild-type GNB1 was sufficient to promote cytokine-independent growth of human TF1 cells. The published structure of GNB1 (Ford et al. Science 1998) reported a small number of residues, including K57, I80 and K89 that mediate interactions with both G-alpha subunits and effector proteins. In fact, affinity purification followed by MS using tagged GNB1 (wild-type, I80T and K89E) demonstrated that, unlike wild-type GNB1, the GNB1 mutants fail to bind distinct Gα subunits. The repertoire of protein interactors, which includes potential G protein effectors, also differed between different GNB1 alleles. Thus, gain-of-function mutations in GNB1 occur across a broad range of hematologic malignancies, modify essential interaction G-protein subunit interactions, can drive in vivo transformation, and activate targetable downstream kinases. Disclosures:Tyner:Incyte Corporation: Research Funding.

CX-4945, An Orally Bioavailable Selective Inhibitor of Casein Kinase 2 (CK2), Exhibits Anti-Tumor Activity in Hematologic Malignancies,

Abstract 3512 Background:CK2 is a highly conserved and constitutively active serine-threonine protein kinase that plays a fundamental role in maintaining cell survival through pro-proliferative, anti-apoptotic and pro-angiogenic signaling. It is comprised of two catalytic (α and α’) and two regulatory (β) subunits.Overexpression and hyperactivation of CK2 has been well described in a variety of human solid tumors, and more recently, in hematologic malignancies including chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), T-cell acute lymphocytic leukemia (T-ALL), and multiple myeloma (MM). CK2 is known to support the viability of cancer cells through increased activation of the downstream PI3K/AKT signaling pathway, which results in cell-cycle progression, and suppression of apoptosis. Inhibition of CK2 has been shown to lead to preferential apoptosis of CLL, AML and myeloma cells. Studies have also demonstrated that CK2 inhibition in MM enhances the cytotoxic effect of chemotherapeutic drugs. Therefore, CK2 appears to be a rational target for novel therapies to treat hematologic malignancies.CX-4945 is a potent and orally bioavailable inhibitor of both isoforms of the CK2 catalytic subunits, and is the first small molecule inhibitor of CK2 to progress to human clinical trials. CX-4945 has been found to be highly selective for CK2 and has also been shown to inhibit phosphorylation of the CK2-specific S129 site of AKT and inhibit activation of the AKT pathway. Anti-tumor activity of CX-4945 has been validated in cancer cell lines and murine xenograft models, and biological activity was observed in early clinical trials with solid tumors and multiple myeloma. However, little is known about its possible effects in leukemias and lymphomas. Therefore, we explored the therapeutic potential of CX-4945 in 224 adult and pediatric patient samples across a wide spectrum of hematologic malignancies including AML, ALL, CLL, CML, CMML and leukemic phase lymphomas. Methods:Fresh primary cells from 224 patients were purified using a Ficoll gradient. Purified cells were then added to wells (5 × 104 per well) containing 10% serum containing media and four serial dilutions of CX-4945 (ranging from 10 nM to 10 μM). Three days after culturing cells in the presence or absence of each drug concentration, we performed a tetrazolium-based cell viability assay (MTS) to evaluate the effect of CX-4945 across a broad spectrum of primary hematologic malignancy samples. The viability data were normalized to untreated controls and used to calculate IC50 values. Results:Of the 7 categories of hematologic malignancies evaluated, all except for 2 (CMML and CML) demonstrated a subset of patients with significantly decreased viability (IC50 < 1 μM) in response to CX-4945 (Table 1).Table 1:IC50 <1 μM by tumor subtype (all samples are adult unless otherwise indicated)Tumor typeNumber samples evaluatedNumber sensitive (Percent sensitive)Median IC50(of sensitive samples)CLL74 total16 (21.6%)415.9 nMAML60 total5 (8.3%)709.9 nM54 adult5 (9.3%)709.9 nM6 pediatric0 (–)–B-ALL58 total3 (5.2%)595 nM23 adult1 (4.3%)908.2 nM35 pediatric2 (5.7%)315 nMCMML13 total0 (–)–T-ALL4 total1 (25%)7.6 nM3 adult1 (33%)7.6 nM1 pediatric0 (–)–Lymphomas11 total3 (27.3%)305.7 nM1 Burkitt1 Burkitt (100%)6 Mantle Cell2 Mantle Cell (33%)2 Diffuse Large Cell1 Follicular1 Marginal ZoneCML4 total0 (–)–3 adult1 pediatricInvestigations evaluating the effect of CK2 on downstream targets such as AKT and other cellular protein kinases, as well as studies exploring CK2 expression in AML are ongoing and will be presented. Conclusions:Our data confirms that CX-4945 demonstrates significant pre-clinical activity across a broad range of hematologic malignancies, and also confirms previous findings that CK2 may play a significant role in the tumorigenesis of certain hematologic malignancies. CX-4945 may be an attractive compound for clinical development, particularly in lymphoid malignancies such as CLL. Future studies will focus on the identification of highly sensitive subsets of cells, further defining the mechanistic details of the CK2 pathway in hematologic malignancies, and exploring the specific features that drive response to CK2 inhibition. Disclosures:Druker:Cylene Pharmaceuticals: Consultancy. Loriaux:Cylene Pharmaceuticals: Research Funding.

Phase I Study of the Aurora B Kinase Inhibitor Barasertib (AZD1152) to Assess the Pharmacokinetics (PK), Metabolism and Excretion in Patients (pts) with Relapsed or Refractory Acute Myeloid Leukemia (AML)

Abstract 2602 Background:Barasertib is a pro-drug that rapidly undergoes phosphatase-mediated cleavage in serum to release barasertib-hQPA (bara-hQPA), a highly potent and selective inhibitor of Aurora B kinase with antitumor activity in a broad range of hematological malignancies including AML (Löwenberg et al. Blood 2009;114:abst 2080; Tsuboi et al. Leuk Res 2011; doi: 10.1016/j.leukres.2011.04.008). Methods:In this open-label, single-arm study (NCT01019161), AML pts received 1200 mg barasertib as a 7-day continuous iv infusion every 28 days. On Day 2 of Cycle 1 only, pts also received 14C-barasertib (250 μCi; 2-h infusion). Blood, urine and feces samples were collected at various time points during Cycle 1 to characterize the PK, metabolic and excretion profiles of barasertib. Results:Of the 6 pts enrolled (median age 63 years [range, 34–74]; 3 females), 5 commenced treatment and completed Cycle 1 and were therefore eligible for analysis. Of the 4 pts evaluable for disease response, 1 achieved a complete response. No new or unexpected safety findings were reported. Maximal plasma concentrations of barasertib and bara-hQPA were achieved by the first scheduled sample, taken 24 h from the start of infusion (SOI). During infusion, the mean concentration of bara-hQPA was approximately 3-fold higher than that of barasertib (Table). Following the end of infusion (EOI), plasma concentrations of barasertib fell rapidly, reaching the limit of quantification (LoQ) by 6 h after EOI, whereas bara-hQPA plasma levels declined in a triphasic manner, with low concentrations still detectable at the final sampling time point (Day 18). Bara-hQPA was extensively distributed to the tissues, with a relatively slow rate of total clearance (Table). Urine concentrations of barasertib were below LoQ at all time points. The mean urine concentrations of bara-hQPA were approximately 4–5 μg/mL during the infusion period, declining rapidly following EOI. The renal clearance (CLR) values for bara-hQPA represent approximately 10% of the total clearance of bara-hQPA from plasma.Overall, 72–82% of radioactivity was recovered, with approximately double the amount recovered in feces (mean = 51%) compared with urine (mean = 27%). The excretion of radioactivity in urine occurred predominantly within 72 h of 14C-SOI; however, there was large inter-patient variability in the rate of recovery of radioactivity in feces. Overall, the main excreta metabolites identified were: bara-hQPA (range, 13.2–33.7%), bara-hQPA N-acetic acid (range, 7.8–10.5%), bara-hQPA desfluoroaniline N-acetic acid (range, 5.1–9.5%), N-formyl or ethoxy bara-hQPA (range, 3.5–9.2%), and bara-hQPA desfluoroaniline (range, 1.6–5.5%). Desfluoroaniline metabolites made up a significantly larger proportion of the metabolites identified in excreta (∼15%) than in plasma (∼2%). The main pathways identified in human metabolism of barasertib were (i) cleavage of the phosphate group to form bara-hQPA, followed by oxidation, and (ii) loss of the fluoroaniline moiety to form bara-hQPA desfluoroaniline, followed by oxidation.Pharmacokinetics of barasertib and bara-hQPA in plasma and urineSampleParameter*Barasertib (n=5)‡Bara-hQPA (n=5)‡PlasmaAUC, ng.h/mL†NC40400 (0.4)AUC(0–t), ng.h/mL†13840 (0.4)40080 (0.4)AUC(0–192 h), ng.h/mL†NC38230 (0.4)CSS, ng/mL†85.6 (0.5)226.8 (0.4)CL, L/hNC31.4 (11.3)t½, hNC66.3 (36.1)λZ, h-1NC0.02 (0.02)VSS, LNC669.1 (344.9)VZ, LNC2805 (1708)UrineAe, mgNC96.9 (47.2)CLR, L/hNC2.85 (1.6)fe, %NC8.1 (3.9)AUC, area under plasma concentration–time curve; CSS, steady-state drug concentration; CL, total clearance; t½, half-life; λZ, slowest disposition rate constant; VSS, volume of distribution (apparent) at steady state; VZ, volume of distribution (apparent) during terminal (λZ) phase; Ae, cumulative amount of unchanged drug excreted; CLR, renal clearance of drug from plasma; fe, fraction of drug excreted into urine; NC, not calculable*All values are arithmetic mean (standard deviation) except where indicated†Values indicate geometric mean (coefficient of variation)‡Due to sampling issues, plasma concentration data were excluded for 1 pt Conclusions:The PK profile of bara-hQPA is similar to previous studies using the same dosing regimen. The majority of clearance of bara-hQPA occurred via hepatic metabolic routes. This study is the first to characterize the metabolic pathway of barasertib in humans. Disclosures:Oliver:AstraZeneca: Employment. D'Souza:AstraZeneca: Employment, Equity Ownership. Pike:AstraZeneca: Employment, Equity Ownership. Stockman:AstraZeneca: Employment, Equity Ownership.

T cells redirected against CD70 for the immunotherapy of CD70-positive malignancies

AbstractT-cell therapy with genetically modified T cells targeting CD19 or CD20 holds promise for the immunotherapy of hematologic malignancies. These targets, however, are only present on B cell–derived malignancies, and because they are broadly expressed in the hematopoietic system, their targeting may have unwanted consequences. To expand T-cell therapies to hematologic malignancies that are not B cell–derived, we determined whether T cells can be redirected to CD70, an antigen expressed by limited subsets of normal lymphocytes and dendritic cells, but aberrantly expressed by a broad range of hematologic malignancies and some solid tumors. To generate CD70-specific T cells, we constructed a chimeric antigen receptor (CAR) consisting of the CD70 receptor (CD27) fused to the CD3-ζ chain. Stimulation of T cells expressing CD70-specific CARs resulted in CD27 costimulation and recognition of CD70-positive tumor cell lines and primary tumor cells, as shown by IFN-γ and IL-2 secretion and by tumor cell killing. Adoptively transferred CD70-specific T cells induced sustained regression of established murine xenografts. Therefore, CD70-specific T cells may be a promising immunotherapeutic approach for CD70-positive malignancies.

Phase I Study of Panobinostat Plus Decitabine In Elderly Patients with Advanced MDS or AML.

AbstractAbstract 1060 Introduction:Panobinostat (LBH589) is pan-deacetylase inhibitor of both histones and nonhistone proteins such as HSP90 and HIF-α which are implicated in leukemogenesis. Panibinostat has demonstrated activity in a broad range of hematologic malignancies including AML with in vitro studies demonstrating synergistic mechanism of action with a number of agents including DNA hypomethylating agents. Methods:We conducted a phase I study of panobinostat plus decitabine in elderly patients with advanced MDS/AML. Patients age ≥ 60 years with advanced MDS (IPSS ≥ 1.5) or AML who had not been previously treated with a hypomethylating agent were eligible for the study. Decitabine 20mg/m2/d IV on days 1–5 was administered with panobinostat po 3x/wk on nonconsecutive days of a 28 day cycle for up to 12 cycles. panobinostat started at 10 mg/d and was escalated to a maximum of 40 mg/day in 5 cohorts using a 3+3 design. The 40 mg dose group was the highest allowed in the study based on anticipated cytopenias from both drugs in an elderly population. Results:Twenty-eight patients (21 AML/7 MDS) with a median age of 71 years (range 60–86), median WBC 12.7 (range 0.9–73.5) were treated in the Phase I study. Twelve of these patients had previously been treated with regimens that included 7+3 or high dose cytarabine (6 pts, 21%) or high dose lenalidomide (6 pts, 21%). The dose of panobinostat was escalated to a maximum of 40 mg 3x/wk. Of the first 27 evaluable patients there were 7/27 (22%) complete responses with 4 of 8 patients in the 30 mg/day cohort achieving a CR (1 CR, 3 CRi). Of the patients with a CRi, two had baseline cytogenetic abnormalities and both experienced disappearance of the abnormality at the time of response. These patients had persistent thrombocytopenia which may be a consequence of treatment rather than the presence of residual leukemia. Side effects included a dose-limiting asthenia (1 pt /each) which occurred in both the 30 mg and 40 mg/day cohorts. Disease progression was the most common reason for discontinuation of study treatment occurring in 8 pts (four of which occurred in the first cycle of therapy). These early cases of disease progression may suggest that “low-dose” therapies in patients should be avoided in patients with hyperproliferative disease. We conclude that the combination of panobinostat plus decitabine can be safely administered to patients with AML/MDS. The response rate observed at higher doses (' 30 mg/d) is encouraging and warrants further investigation. Based on this phase I data which demonstrates encouraging evidence of clinical activity for the combination, a phase II cohort using a dose of panobinostat 40 mg po 3x/wk is currently being enrolled. Disclosures:Uy:Novartis: Research Funding. Off Label Use: Panobinostat for MDS/AML. Abboud:Novartis: Honoraria. Vij:Novartis: Honoraria; Eisai: Speakers Bureau. Westervelt:Novartis: Speakers Bureau.

MK-0457, a Novel Multikinase Inhibitor, Has Activity in Refractory AML, Including Transformed JAK2 Positive Myeloproliferative Disease (MPD), and in Philadelphia-Positive ALL.

Background. MK-0457 (VX-680) is a small-molecule inhibitor of aurora kinases A, B, and C, FLT3, BCR-ABL, and JAK2. MK-0457 exhibits nanomolar level broad-spectrum preclinical anti-tumor activity. Specifically, MK-0457 inhibits proliferation of patient-derived AML cells in vitro, and improves survival of a Ba/F3 FLT3 ITD murine model of systemic AML. A Phase I study of MK-0457 is being conducted in patients with a broad range of hematological malignancies, including acute myeloid and lymphoid leukemias.Methods. After IRB approval, 15 consenting patients with relapsed/refractory AML and ALL, ECOG performance status ≤ 2, and adequate organ function were enrolled using a standard dose escalation scheme with 3 patients/dose level until dose-limiting toxicity (DLT), followed by 6 patients/level. MK-0457 was administered by continuous 5-day intravenous infusion every 2 to 3 weeks. DLT was defined as grade 3 or higher non-hematologic toxicity during cycle 1. Pharmacokinetics (PKs) were collected pre-dose through 168 h and analyzed for MK-0457 by HPLC/mass spec.Results. Thirteen patients with AML and 2 patients with ALL were enrolled at 8, 12, 20, 24, and 28 mg/m2/hr. Among AML patients, two had a diploid karyotype and the remainder had complex unfavorable cytogenetic abnormalities. Two AML patients had a prior JAK2-positive MPD, transformed to AML, and then received MK-0457 as their 1st AML treatment. One AML patient received MK-0457 as first salvage, three as second salvage, and the remainder as salvage attempt three or higher. Both ALL patients failed prior multiagent chemotherapy; one of these patients had Philadelphia (Ph)-positive ALL and progressed after prior BCR-ABL inhibitor therapy, including dasatinib. The latter patient carried the BCR-ABL inhibitor resistance mutation, T315I. Four of 5 AML patients without baseline grade 3/4 myelosuppression in one cell line developed grade 4 neutropenia, including both JAK2-positive AML patients. Normalization of the platelet count during cycle one occurred in one MPD patient with thrombocytosis at baseline (~800 x 103/mL); Grade 3 thrombocytopenia occurred during cycle one in the other MPD patient with a normal baseline. The Ph+-ALL patient had eradication of peripheral blood blasts at the end of 2 cycles of therapy. No MK-0457 attributable extramedullary grade 2 or above adverse events were seen. Mild hair thinning was seen in some patients at dose levels 20 mg/m2/hr and above. Preliminary PK analysis showed dose-dependent linearity at steady state, with a biexponential decay at the end of infusion characterized by a rapid a decay followed by a slower b decay (t1/2 10–20 hrs).Conclusions. MK-0457 at well tolerated doses achieves myelosuppression in refractory AML and ALL patients. Its activity in JAK2+ transformed AML patients may be partially attributable to JAK2 inhibitory activity. The role of aurora kinase inhibition in the above responses is not yet established. As neither maximum tolerated dose nor dose limiting toxicity has been defined to date, dose finding on this Phase I study of MK-0457 is on-going in the acute leukemia population with 36 mg/m2/hr as the current dose level under investigation.

Trisomy 4 may occur in a broad range of hematologic malignancies

Trisomy 4 is an uncommon numerical chromosomal aberration in acute leukemia. We describe three cases of trisomy 4, occurring in two patients with acute myeloid leukemia (M1 and M5a) and in one patient with T-cell acute lymphoblastic leukemia. Our results suggest that trisomy 4 may occur in a broader range of hematologic malignancies than previously described.