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Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

Proteasome inhibitors, a novel class of chemotherapeutic agents, enhance the antitumor efficacy of anthracyclines in vitro and in vivo. We therefore sought to determine the maximum tolerated dose (MTD) and dose-limiting toxicities of bortezomib and pegylated liposomal doxorubicin (PegLD). Bortezomib was given on days 1, 4, 8, and 11 from 0.90 to 1.50 mg/m2 and PegLD on day 4 at 30 mg/m2 to 42 patients with advanced hematologic malignancies. Grade 3 or 4 toxicities in at least 10% of patients included thrombocytopenia, lymphopenia, neutropenia, fatigue, pneumonia, peripheral neuropathy, febrile neutropenia, and diarrhea. The MTD based on cycle 1 was 1.50 and 30 mg/m2 of bortezomib and PegLD, respectively. However, due to frequent dose reductions and delays at this level, 1.30 and 30 mg/m2 are recommended for further study. Pharmacokinetic and pharmacodynamic studies did not find significant drug interactions between these agents. Antitumor activity was seen against multiple myeloma, with 8 of 22 evaluable patients having a complete response (CR) or near-CR, including several with anthracycline-refractory disease, and another 8 having partial responses (PRs). One patient with relapsed/refractory T-cell non-Hodgkin lymphoma (NHL) achieved a CR, whereas 2 patients each with acute myeloid leukemia and B-cell NHL had PRs. Bortezomib/PegLD was safely administered in this study with promising antitumor activity, supporting further testing of this regimen.

We have shown that thalidomide (Thal) and its immunomodulatory derivatives (IMiDs), proteasome inhibitor PS-341, and As2O3act directly on multiple myeloma (MM) cells and in the bone marrow (BM) milieu to overcome drug resistance. Although Thal/IMiDs, PS-341, and As2O3inhibit nuclear factor (NF)-κB activation, they also have multiple and varied other actions. In this study, we therefore specifically address the role of NF-κB blockade in mediating anti-MM activity. To characterize the effect of specific NF-κB blockade on MM cell growth and survival in vitro, we used an IκB kinase (IKK) inhibitor (PS-1145). Our studies demonstrate that PS-1145 and PS-341 block TNFα-induced NF-κB activation in a dose- and time-dependent fashion in MM cells through inhibition of IκBα phosphorylation and degradation of IκBα, respectively. Dexamethasone (Dex), which up-regulates IκBα protein, enhances blockade of NF-κB activation by PS-1145. Moreover, PS-1145 blocks the protective effect of IL-6 against Dex-induced apotosis. TNFα-induced intracellular adhesion molecule (ICAM)-1 expression on both RPMI8226 and MM.1S cells is also inhibited by PS-1145. Moreover, PS-1145 inhibits both IL-6 secretion from BMSCs triggered by MM cell adhesion and proliferation of MM cells adherent to BMSCs. However, in contrast to PS-341, PS-1145 only partially (20–50%) inhibits MM cell proliferation, suggesting that NF-κB blockade cannot account for all of the anti-MM activity of PS-341. Importantly, however, TNFα induces MM cell toxicity in the presence of PS-1145. These studies demonstrate that specific targeting of NF-κB can overcome the growth and survival advantage conferred both by tumor cell binding to BMSCs and cytokine secretion in the BM milieu. Furthermore, they provide the framework for clinical evaluation of novel MM therapies based upon targeting NF-κB.

Increasing evidence suggests that tissue transglutaminase (tTGase; type II) is externalized from cells, where it may play a key role in cell attachment and spreading and in the stabilization of the extracellular matrix (ECM) through protein cross-linking. However, the relationship between these different functions and the enzyme's mechanism of secretion is not fully understood. We have investigated the role of tTGase in cell migration using two stably transfected fibroblast cell lines in which expression of tTGase in its active and inactive (C277S mutant) states is inducible through the tetracycline-regulated system. Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin. Both forms of the enzyme could be detected on the cell surface, but only the clone overexpressing catalytically active tTGase deposited the enzyme into the ECM and cell growth medium. Cells overexpressing the inactive form of tTGase did not deposit the enzyme into the ECM or secrete it into the cell culture medium. Similar results were obtained when cells were transfected with tTGase mutated at Tyr274(Y274A), the proposed site for the cis,transpeptide bond, suggesting that tTGase activity and/or its tertiary conformation dependent on this bond may be essential for its externalization mechanism. These results indicate that tTGase regulates cell motility as a novel cell-surface adhesion protein rather than as a matrix-cross-linking enzyme. They also provide further important insights into the mechanism of externalization of the enzyme into the extracellular matrix.

ABSTRACTSome retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9Gagproteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecipitation and time course immunoblot analyses showed that proteasome inactivation slightly decreased virus release (at most a twofold effect), while it did not affect Gag processing. These results contrast with those obtained with other viruses which are sensitive to these inhibitors. This suggests that, although its Gag is monoubiquitinated, the requirements for EIAV release are somewhat different from those for retroviruses that are sensitive to proteasome inhibitors.

Over the last decade, the critical role of the proteasome in cell‐cycle regulation has become increasingly apparent. The proteasome, a multicatalytic protease present in all eukaryotic cells, is the primary component of the protein degradation pathway of the cell. By degrading regulatory proteins (or their inhibitors), the proteasome serves as a central conduit for many cellular regulatory signals and, thus, is a novel target for therapeutic drugs. PS‐341 is a small molecule that is a potent and selective inhibitor of the proteasome. In vitro and mouse xenograft studies of PS‐341 have shown antitumor activity in a variety of tumor types, including myeloma, chronic lymphocytic leukemia, prostate cancer, pancreatic cancer, and colon cancer, among others. Although PS‐341 rapidly leaves the vascular compartment, a novel pharmacodynamic assay has shown that inhibition of proteasome—the biologic target—is dose dependent and reversible. These studies provided the rationale for a twice‐weekly dosing schedule employed in ongoing clinical studies. Phase I trials in a variety of tumor types have shown PS‐341 to be well tolerated, and phase II trials in several hematologic malignancies and solid tumor types are now in progress. Efficacy and safety data from the most advanced of these, a phase II multicenter trial in myeloma, will be available in early 2002.

Adoptive transfer of NK cells is a promising immunotherapeutic modality, however limited NK cell persistence and proliferation in vivo have historically been barriers to clinical success. Nicotinamide (NAM), an allosteric inhibitor of NAD-dependent enzymes, has been shown to preserve cell function and prevent differentiation in ex vivo culture of NK (NAM-NK) and other cells. Clinical responses were observed in a Phase 1 trial of NAM-NK (GDA-201) in patients with refractory non-Hodgkin lymphoma (Bachanova, et. al., Blood 134:777, 2019). We now use transcriptional and metabolic profiling to characterize the mechanisms underlying the activity of NAM-NK.

Evolutionary changes are described in plasmid‐containing strains isolated after approximately 800 generations of growth in glucose‐limited chemostat culture. The reproductive fitness increased dramatically over this period. Genetic changes associated with the increases in fitness were localized to both the bacterial and the plasmid chromosomes. In addition, some of the genetic changes on the bacterial and the plasmid chromosomes interact to minimize the deleterious effect of the plasmid. Thus, the changes observed may be considered coevolutionary. Reductions in the deleterious effects of the plasmid were shown to be associated with a decrease in plasmid copy number and an increase in the rate of segregational loss of the plasmid.

It is generally accepted from the darwinian theory of evolution that a progressive increase in population adaptation will occur in populations containing genetic variation in fitness, until a stable equilibrium is reached and/or the additive genetic variation is exhausted. However, the theoretical literature of population genetics documents exceptions where mean population fitness may decrease in response to evolutionary changes in gene frequency, due to varying selective coefficients1–4, sexual selection5,6or to epistatic interactions between loci7. Until now, no examples of such exceptions have been documented from fitness estimates in either natural or experimental populations. We present here direct evidence that, as a result of epistatic interactions between adaptive mutations, mean population fitness can decrease in asexual evolving populations of the yeast Saccharomyces cerevisiae.

An approach is described for the estimation of the number of rare variants in the population from the number in a sample drawn at random from the population. This quantity is used to derive an estimate of the mutation rate. The data required are the number of rare variants in the sample and the distribution of offspring within a population of well-defined size with little or no immigration. Application of this approach to data on 28 loci assayed in the Yanamamo, a tribe of South American Indians, yields an average mutation rate of 0.1 ∼ 0.2 × 10-5. Determination of this figure is subject to several assumptions concerning the nature of the rare variants and the structure of the population. Violation of these assumptions will generally result in the underestimate of the true mutation rate.

Chimpanzees were challenged with HIV-1IIIBwhile receiving a short regimen of nevirapine (Viramune), a nonnucleoside inhibitor of HIV-1 reverse transcriptase. The untreated, control chimpanzee developed an infection characterized by seroconversion, viremia in peripheral blood mononuclear cells (PBMCs), and plasma positive for viral RNA. In contrast, the three nevirapine-treated chimpanzees remained negative for all viral markers with the exception of nested polymerase chain reaction (PCR) analysis of PBMCs for viral DNA. Although PBMCs from the three nevirapine-treated chimpanzees tested intermittently positive for viral DNA, this PCR signal disappeared and remained negative for the final five months of the study. These data indicate that orally administered nevirapine provided protection from HIV-1 infection in the chimpanzee model.

Adaptive mutations are shown to have a higher frequency of fixation in evolving diploid than in haploid populations of the yeast Saccharomyces cerevisiae, providing direct evidence that it may be an evolutionary advantage to be diploid.

Interactions between various genotypes were studied in four varieties of barley, four varieties of wheat, and eight barley genotypes which had survived up to 18 generations of mutual selection in a heterogeneous population. The results showed that first-order interactions between genotypes from the heterogeneous barley population were much larger than those between the varieties of wheat and barley. Natural selection therefore appears to preserve genotypes which interact synergistically. Deterministic and stochastic simulations predicted fixation of the variety with the highest pure-stand reproductive value in populations of the wheat and barley varieties. However, they predicted the development of stable multigenotypic associations in populations formed from the eight genotypes isolated from the heterogeneous barley population. The effect of random variation in the reproductive values was to decrease the time to fixation when there was no stable nontrivial equilibrium; however, when such an equilibrium existed, stochastic variation increased genetic diversity. The significance of intergenotypic interactions in maintaining genetic diversity was discussed.

Lodie: Gamida Cell: Employment, Equity Ownership. Adams:Gamida Cell: Employment, Equity Ownership. Yackoubov:GAMIDA CELL: Employment, Other: unexecuted shares of the company . Peled:Gamida Cell: Employment, Equity Ownership.

Historical efforts at expansion of umbilical cord blood (UCB) derived CD34+ hematopoietic stem cells (HSCs) ex vivowith cytokines yielded large numbers of progenitors for transplantation but impaired their long-term engraftment ability. We used nicotinamide (NAM), an allosteric inhibitor of NAD-enzymes, to create omidubicel, an investigational cell therapy designed to improve the expansion of CD34+ HSCs for bone marrow transplant. A Phase 1/2 clinical study of omidubicel in patients with high-risk hematologic malignancies showed rapid neutrophil engraftment and a more favorable immune reconstitution profile in patients compared to historical controls.1We hypothesized that NAM treatment maintains the stemness and engraftment potential of omidubicel, which is associated with clinical benefit.2

No relevant conflicts of interest to declare.

Introduction: IPI-504 is a novel, water-soluble Heat Shock Protein 90 (Hsp90) inhibitor. It is the chemical reduction product of 17-AAG, a well-characterized Hsp90 inhibitor. 17-AAG is currently in clinical trials, but suffers from very poor aqueous solubility and must be administered to patients in either suboptimal DMSO formulations, or newer cremophor or emulsion formulations. In contrast, IPI-504 exists as a hydrochloride salt which is soluble in water in excess of 200 mg/mL. Therefore, IPI-504 is 4000-fold more soluble than 17-AAG. It has previously been shown that IPI-504 inter-converts with 17-AAG and exists in a pH and enzyme-mediated dynamic redox equilibrium which has been observed in human clinical trials as well. In preclinical animal studies, IPI-504 has demonstrated in vitro and in vivo anti-tumor effects in a variety of cancers including xenograft and orthotopic models of multiple myeloma (MM). Methods: An open-label phase I dose-escalation trial of IPI-504 infused on days 1, 4, 8 and 11 of a 21 day cycle is being conducted in patients (pts) with relapsed/refractory MM. IPI-504 is administered to pts in 250cc of normal saline over thirty minutes. The primary objectives of the study are to determine the maximum tolerated dose (MTD) and characterize the pharmacokinetic (PK) and pharmacodynamic (PD) marker effects of IPI-504. Levels of Hsp70 protein were measured as a marker of IPI-504’s biologic activity in peripheral blood leukocytes. Toxicity is evaluated by NCI CTCAE (v3.0) and response by EBMT. An accelerated dose titration design was employed with dose escalation of 100% for the first three dose levels with 1 pt / cohort. The first three pts received 22.5, 45 and 90 mg/m2 respectively. Per protocol, once the 150 mg/m2 level was achieved, dose escalation converted to a modified Fibonacci schema with 3 pts per cohort. Subsequently dose escalation occurred in increments of 66%, 50%, 33% and 25%. Results: Currently, 15 pts (11 female / 4 male), average age 61.3 years, have been treated. IPI-504 has been well-tolerated at doses ranging from 90 to 300 mg/m2. Patients continue to be enrolled at the 400 mg/m2 dose level. Dose limiting toxicities (DLTs), defined as grade 4 or greater hematologic toxicity and/or grade 3 or greater non-hematologic toxicity within the first 21 days of treatment, have not been observed to date. Dose escalation will continue until a recommended phase 2 dose is identified. Analysis of PK, safety, tolerability, and biologic response to IPI-504 is ongoing. The above support the planning of further clinical exploration of IPI-504 including combination with agents such as bortezomib that have proven synergistic in animal models of MM.

IPI-504 is a novel, water-soluble Heat Shock Protein 90 (Hsp90) inhibitor. It is the chemical reduction product of 17-AAG, a well-characterized Hsp90 inhibitor. 17-AAG is currently in clinical trials, but suffers from very poor aqueous solubility and must be administered to patients in either suboptimal DMSO formulations, or newer cremophor or emulsion formulations. IPI-504, however, exists as a hydrochloride salt which is soluble in water in excess of 100 mg/mL. It has previously been shown that IPI-504 inter-converts with 17-AAG and exists in a pH and enzyme-mediated dynamic redox equilibrium. When IPI-504 or 17-AAG is administered to mice, rats, or monkeys a dynamic equilibrium occurs in vivo in which both species are present in plasma as well as tissue. Thus, IPI-504 recapitulates all of the Hsp90 inhibitory properties and biological activity of 17-AAG without its formulation liabilities. In preclinical studies IPI-504 has demonstrated in vitro and in vivo anti-tumor effects in a variety of cancers including xenograft and orthotopic models of multiple myeloma. Therefore an open-label phase I dose-escalation trial of IPI-504 infused on days 1, 4, 8 and 11 of a 21 day cycle is being conducted in pts with relapsed/refractory MM. IPI-504 is infused in 250cc of normal saline over thirty minutes. Pts receive IPI-504 for one cycle of treatment. The primary objectives of the study are to determine the maximum tolerated dose (MTD) and characterize the pharmacokinetic (PK) and pharmacodynamic (PD) marker effects of IPI-504. An accelerated titration design was utilized during the first three dose levels. Dose limiting toxicity (DLT) is defined as grade 4 or greater hematologic toxicity and/or grade 3 or greater non-hematologic toxicity within the first 21 days of treatment. Results demonstrate that the in vivo dynamic equilibrium of IPI-504 and 17-AAG is detectable in human plasma PK samples and the water-based formulation is well-tolerated. Data on PK, dose proportionality, safety, tolerability, PD markers, and anti-tumor activity of IPI-504 will be reported. In conclusion, clinical evaluation of this agent is ongoing to define the safety, tolerability and potential activity of IPI-504 to treat pts with advanced MM. These results may help clarify the effects of Hsp90 inhibition using a water-soluble, ansamycin-based Hsp90 inhibitor without the confounding effects of DMSO-based formulations.

We have previously shown that inhibitors of the hsp90 molecular chaperone (including geldanamycin, 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and other members of the ansamycin family) potently induce growth arrest and apoptosis of a large panel of drug-sensitive and -resistant MM cell lines, as well as tumor cells freshly isolated from patients with relapsed refractory MM; and sensitize these cells to other pro-apoptotic anti-tumor agents. While multiple phase I clinical trials have shown that biologically active doses of 17-AAG can be administered without significant hsp90-related toxicities, the insolubility of this compound in most conventional clinical solvents, as well as the practical limitations of DMSO-based formulations that were used in the original clinical trials have generated the need to develop more effective and practical approaches to administer 17-AAG to patients. Herein we describe the in vitro and in vivo pre-clinical profile of IPI-504, a novel analog of 17-AAG, which is soluble in aqueous formulations and can bypass key limitations of the DMSO-based formulations for administration of 17-AAG. Our in vitro studies show that IPI-504 has anti-tumor activity against a broad panel of primary MM tumor cells as well as MM cell lines (including cells resistant to cytotoxic chemotherapeutics, proteasome inhibitor bortezomib, thalidomide or its immunomodulatory thalidomide derivatives, and/or Apo2L/TRAIL). Based on hierarchical clustering analyses, logistic and linear regression models, we observed that the profiles of drug sensitivity of MM cells to IPI-504 were consistent with the profiles of sensitivity to 17-AAG. Similarly, IPI-504 triggered a constellation of molecular sequelae that were consistent with hsp90 inhibition by 17-AAG, including suppression of cell surface expression and down-stream signaling (via PI-3K/Akt and Ras/Raf/MAPK) of receptors for IGF-1 and IL-6; decreased intracellular levels of several key kinases, including Akt, Raf, IKK-α; suppressed expression of several intracellular anti-apoptotic proteins (e.g. FLIP, XIAP, cIAP2); leading to tumor cell sensitization to other pro-apoptotic agents (e.g. cytotoxic chemotherapy or PS-341). Importantly, in our mouse model of diffuse MM bone lesions in SCID/NOD mice, IPI-504 (50 mg/kg, i.v. twice weekly) was able to prolong the survival of mice vs. vehicle-treated mice (p<0.01, log-rank test), without significant treatment-related toxicities. These results indicate that hsp90 inhibitors have significant anti-MM activity in vivo, which, coupled with our ex vivo mechanistic and molecular profiling studies, have provided the framework for upcoming clinical trials of this novel class of agents in patients with MM.

IPI-504 is a novel inhibitor of Hsp90 based on the geldanamycin pharmacophore. When placed in rat, monkey, and human blood, IPI-504 rapidly converts to the known and well-studied compound 17-allylamino-17-demethoxy-geldanamycin (17-AAG). 17-AAG is the subject of multiple clinical trials for the treatment of hematologic and solid tumors. However, 17-AAG suffers from poor aqueous solubility necessitating the use of sub-optimal formulations to deliver this agent to patients. IPI-504 is over 1000-fold more soluble than 17-AAG in aqueous solution. In vitro, both 17-AAG and IPI-504 bind tightly to, and selectively inhibit Hsp90 derived from cancer cells. The cytotoxic effect of IPI-504, as well as its ability to stimulate the degradation of Hsp90 client proteins and increase the intracellular levels Hsp70, were monitored in two human multiple myeloma cells lines (RPMI-8226 and MM1.S). The effects of IPI-504 were compared to 17-AAG. We demonstrate that the actions of IPI-504 are bioequivalent to 17-AAG and that both compounds induce apoptosis in these cells and stimulate the degradation of HER2 and c-Raf. In addition, both agents stimulate Hsp70 protein levels. In all cases the EC50s are virtually the same for both molecules (~200–400 nM). Furthermore, IPI-504 inhibits the secretion of immunoglobulin light chain from the RPMI-8226 multiple myeloma cells (EC50 ~300 nM). Importantly, IPI-504 is active in tumor xenograft models of multiple myeloma. The data indicate that active metabolites of IPI-504 accumulate in these xenografts long after these metabolites are cleared from the plasma compartment, suggesting that they preferentially accumulate in tumor cells based on their increased affinity to Hsp90 derived from tumor cells. In conclusion, we have developed IPI-504 as a novel, potent inhibitor of Hsp90 with greatly increased solubility over 17-AAG, and that IPI-504 is an active anti-tumor agent in vitro and in vivo.

The dipyridodiazepinone human immunodeficiency virus type 1 (HIV-1)-specific reverse transcriptase (RT) inhibitor BI-RG-587was tested for its ability to inhibit HIV-l replication in both acutely and chronically infected cell lines. The ability of BI-RG-587 to inhibit steps in the virus replicative cycle other than reverse transcription was also assessed. BI-RG-587 was found to be a potent inhibitor of HIV-1 replication in acutely infected cells (50% inhibitory concentration [IC50] = 37.2 nM), and the sensitivity and kinetics of that inhibition was similar to the known RT inhibitor zidovudine (AZT). Even at lOO × IC50, BI-RG-587 had no effect on gp120/CD4 interaction, syncytia formation, or envelopeglycoprotein processing. In addition, no inhibition ofviral replication or protein production was noted in a chronically infected cell line that produces viral products in an RT-independent manner. Finally, no inhibition of acute HIV-2 replication was noted, even with very high (2500 × IC50 for HIV-1) concentrations of BI-RG-587. These results demonstrate that BI-RG-587 is a potent inhibitor of HIV-1 replication and that this inhibition occurs at the point of reverse transcription.